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Loading E-Gel® EX Agarose Gels

1. sese 52 H repeloi iia orelliai e t 54 iii Electrophoresis of E Gel EX Agarose Gels eene netten tnit inanes 56 Sample Preparation for E Gel EX Agarose Gels eee eese eet tette teint tntnt ntn ne 56 Preparing RNA Samples for E Gel EX Agarose Gels setas aes Seis ess Eu UR I 58 Loading B Gel EX Agarose Gels ones isesto tinet dta tied AR 59 Rione Bel GA AROSE GOlS id uieie E Stu tabta ues N 61 Masualizins B Gel EX Agarose Gelsissse teet itae ento tatit d dadas oos edet o aded iras iud 63 Opening an E Gel EX Agarose Gel Cassette ipa Quad he Duoidt mel uid dude atcur du 64 Results using E Gel EX Aearose Gels i etti n rat D t ip d beo la Eae ae 66 Results using E Gel EX Agarose Gels for RNA Samples sse 67 Troubleshoot no M EE E 68 DNA Purification Using E Gel CloneWell Agarose Gels eene 70 Sample Preparation for E Gel CloneWell Agarose Gels eene 70 Loading E Gel CloneWell Agarose Gels iscesetestoesisee baee boot iot ova tH Re oae EUH Reg IRE CoU ARIES HERE ERROR pe 72 Running F GelCloneWel anid Collecting DN Assassinio a diuo 75 Visualizing E Gel CloneWell Agarose Gels eene tnt tnt teint tntnt intus 79 Results using B Gel CloneWell Agarose Gels nA anat ns RA TRE RETECU AD RU RES RA UNE 80 MTOUDIESH OO Ung e 81 DNA Purification Using E
2. Press and release the power button Automatic Automatic Press and release the power button during the run Press and release the power button Continuous beeping for 2 minutes followed by a single beep every minute Continuous beeping for 2 minutes followed by a single beep every minute No light if a cassette is not inserted or red light if a cassette is inserted Steady red Steady green Flashing red until the time button is pressed Alternating red and green With gel cassette in steady red Without gel cassette no light Steady red Default time setting 12 minutes or last time setting Count down time Negative time display 00 to 19 minutes Negative time display 00 to 19 minutes Flashing time display Last time setting Continued on next page E Gel 96 Mother Daughter Base Quick Reference Guide Continued Mode Action Sound Light Digital Display Restart after Press and Steady green Count down a manual release the time stop power button Return to Press and hold With gel cassette Last time Ready mode the power in steady red setting after a button Without gel manual stop cassette no light Failure Remove the Rapid Steady red gel cassette beeping from the base No cassette Last time setting Time setting Press and release the increases by time button 1 minute increments Press and hold the time increases button
3. 100 For optimal results load each E Gel 48 and 96 gel within 15 minutes of removing the gel from the plastic pouch and run the gel within 15 minutes of loading If a gel has been out of its plastic pouch for more than 15 minutes you must use the Two Step Loading method described on page 126 Do not pre run E Gel 48 and 96 gels Store and run E Gel agarose gels at room temperature The recommended program for E Gel is EG and the run time for E Gel 48 1 and 2 gels is 20 minutes E Gel 48 4 is 17 minutes and E Gel 96 gels is 12 minutes Alternatively E Gel 96 gels can be run using the EP program with a 6 minute run time though in some cases this may result in a slight reduction of run quality You will need to select an appropriate program on the base prior to inserting a gel into the base Note If you had previously set the E Base to the desired program or set the time the last used program or time is displayed 1 Plug the Mother E Base into an electrical outlet using the plug on the base If using Daughter E Base connect the Daughter E Base to a Mother E Base or to another Daughter E Base connected to a Mother E Base The display will show EP default or last program used EG or EP Select the appropriate program based on the gel Program Gel Type Run Parameters EG E Gel 96 Time 12 minutes EP E Gel 96 Time 6 minutes EG E Gel 48 1 and 2 Time 20 minutes EG E Gel 48
4. Gel Volume 50 ml Gel Percentage 1 and 2 Well Depth 3mm Well Opening 3 8 mm x 1 8 mm Well Bottom 3 3 mm x 1 1 mm Running Distance 16 mm one well to the next Space between Wells 9mm The wells of the E Gel 96 cassette are compatible with a multichannel pipettor or 8 12 or 96 tip robotic loading devices Continued on next page 2J Product Specifications Continued E Base Specifications E Gel iBase Specifications The specifications for Mother E Base and Daughter E Base are listed below Dimensions 14 6 cm x 15 cm x 5 3 cm Weight Mother E Base 370 g Daughter E Base 271g Safety Double Insulation UL listed and CE certified Temperature Ambient 15 C to 40 C Built in Features Digital time display 00 99 minutes alarm light LED The SBS Society for Biomolecules Screening standard 96 well plate format of the E Base fits on most robotic platforms allowing the loading and electrophoresis of gels on the E Base directly on the robot The specifications for E Gel iBase are listed below Dimensions 18 4 cm x 11 cm x 5 75 cm Weight 500 g Safety UL listed and CE certified Temperature Ambient 15 C to 40 C Built in Features Alarm light LED LCD Display E Gel Safe Imager Real time Transilluminator Specifications 28 The specifications for the E Gel Safe Imager Real time Transilluminator are listed below Viewing surface dimensions 62 x 77 mm Case dimen
5. iBase Power System into an electrical outlet using the adaptor plug on the base Open the package and remove the gel Do not remove the comb until you start loading the samples next page Slide the cassette into the two electrode connections on the E Gel iBase Power System Press on the left side of the cassette to secure it into the iBase The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED light will illuminate a steady red to show that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the program PRE RUN 2 min using the Up Down A V buttons Press the Go button to pre run the gel The LED light changes to green light to indicate that the cassette is Run the program PRE RUN 2 min in the pre run mode After two minutes pre run stops automatically as indicated by a red light and a beeping sound Continued on next page 73 Loading E Gel CloneWell Agarose Gels Continued Loading Samples 74 After you have pre run the gel load the samples within 15 minutes as described below All wells in the gel must contain sample or water Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Remove the combs from the top and bottom row of the E Gel CloneWell
6. illuminates on the base when the cassette is correctly base inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Power System Poor resolution Sample is overloaded Do not load more than 200 ng of sample DNA per band ia iia of High salt concentration Dilute your high salt samples as described on page 31 ands Aberrant pre run step Be sure to pre run the gel but do not exceed 2 minutes Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 15 minutes of sample immediately after sample loading loading Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current time or high current during resulting in poor band migration or a melted gel Do the run not run the gel longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 40 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well from the we
7. E Gel Safe Imager Real time Transilluminators passes through a blue filter producing a single intensity signal at approximately 480 nm effective for the excitation of SYBR Safe DNA gel stain the proprietary stain in E Gel EX and E Gel i SizeSelect gels and many of 350 Ax 450 5B 5E ex our other nucleic acid and W avelength nm protein stains such as SYBR Gold SYBR Green I and II SYPRO Ruby SYPRO Orange and Coomassie Fluor Orange S un un Tul Em Unlike UV transilluminators the E Gel Safe Imager Real time Transilluminator does not produce UV light and does not require UV protective equipment during use Blue light transillumination also results in dramatically increased cloning efficiencies compared to UV transillumination The E Gel Safe Imager Real time Transilluminator cannot be used for viewing ethidium bromide stained gels Continued on next page 23 Safe Imager Blue Light Transilluminators Continued Safety Information for the E Gel Safe Imager Real time Transilluminator Viewing Gels with the E Gel Safe Imager Real time Transilluminator 24 The E Gel Safe Imager Real time Transilluminator is an electrical device e Never touch the power cord or outlet with wet hands e Do not use this device in damp areas or while standing on damp floors e Do not attempt to open the E Gel Safe Imager Real time Transilluminator The E Gel Safe Imager R
8. No IR coating on camera when using an UV system Used a UV light source to visualize DNA Solution Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button or Go button for 2 seconds to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase E Gel Base and E Gel PowerBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page 123 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel with SYBR Safe empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use a blue light transilluminator such as the Safe Imager Blue Light Transilluminator or E Gel Safe Imager Real time Transilluminator see page x 55 Electrophoresis of E Gel EX Agarose Gels Sample Preparation for E Gel EX Agarose Gels Introduction E Gel EX agarose gels are pre cast 1 and 2 gels in the E Gel format with 11 wells and a novel openable format The gels contain a proprietary fluorescent nucleic acid stain excitation 490 nm emission 520 nm that can be viewed with blue light which minimizes DNA damage and allows detection down to 1 ng band of DNA For optimal results using E Gel EX agarose gels follow the guidelines for prep
9. To open the E Gel cassette for staining excision of DNA fragments or for blotting see page 9 for details 39 Results with E Gel Single Comb Gels Introduction Results obtained using single comb or double comb E Gel gels are shown below and on the following pages All gels were photographed using a Kodak EDAS120 system You can also use a mini transilluminator to view the bands Note You may vary the amount of markers loaded to improve photography of the gel 0 8 Single Comb Results obtained using a 0 8 E Gel gel are shown below using 20 ul per lane Gel Digestion of pUC18 with Pst I lane 5 linearizes the plasmid 2 7 kb Digestion of pcDNA 3 1 5 4 kb with Nco I lane 3 yields 3 fragments 735 bp 1 4 kb and 3 3 kb 0 8 Agarose GP M UU LE 1 Oo COON DD OFF amp W rH me mM C N Lane Sample High DNA Mass Ladder 200 ng 500 bp DNA Ladder 620 ng pcDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng 1 Kb Plus DNA Ladder 300 ng 1 Kb Plus DNA Ladder 300 ng 3 kb PCR fragment 4 kb PCR fragment 5 kb PCR fragment 500 bp DNA Ladder 620 ng High DNA Mass Ladder 200 ng 1 2 Single Comb Results obtained using a 1 2 E Gel gel are shown below using 20 ul per lane Gel Digestion of pUC18 and pcDNA 3 1 are as described above ote Agarose GP 123 45 6 7 E 8 1 12 40 Lane LO COON D OFF WN m mM C N Sample High DNA Mass Ladder
10. WON A FT i WN E ee Ne oO Continued on next page 53 Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel with SYBR Safe agarose gels No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before cassette the specified expiration date E Gel with SYBR Safe Remove cassette and reinsert a steady red light cassette is not inserted illuminates on the base when the cassette is correctly properly into a base inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase and PowerBase Poor resolution Sample is overloaded Do not load more than 200 ng of sample DNA per band ia iia of High salt concentration Dilute your high salt samples as described on page 31 ands Aberrant pre run step Be sure to pre run the gel but do not exceed 2 minutes Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed F
11. as a size reference marker Collect the band of interest when the two bands in the 50 bp ladder that bracket the band of interest in size are just beginning to enter for the larger marker and exit for the smaller marker the collection well in the marker lane Run Time Estimation Band Size 50 bp 100 bp 150 bp 200 bp 300 bp 400 bp 500 bp 650 bp 800 bp 1000 bp Run Time to Reference Line 8 5 10 minutes 9 10 5 minutes 10 11 5 minutes 11 12 5 minutes 12 14 minutes 13 15 minutes 14 5 16 5 minutes 16 18 minutes 17 5 19 5 minutes 18 5 20 5 minutes Time from Reference Line to Collection Well 0 5 1 minute 0 5 1 minute 0 5 1 minute 0 5 1 5 minute 0 5 1 5 minute 0 5 1 5 minute 0 5 1 5 minute 1 1 5 minute 1 2 minute 1 2 minute The run times indicated are estimates Some bands in different wells may Note migrate differently as DNA fragment size DNA quantity and salt content may slightly affect migration rates 85 Loading E Gel SizeSelect Agarose Gels Introduction After you have prepared your samples you are ready to proceed with electrophoresis Instructions are provided below to load E Gel SizeSelect agarose gels using the E Gel iBase Installing iBase If using the iBase Power System without an E Gel Safe Imager Connect the Power System cord with the transformer to the power inlet of the iBase and plug the other Alone end of the power cord into an electrical outlet Use only
12. documentation The E Editor 2 02 software can be downloaded for free from our Website at www invitrogen com egels System The High Throughput E Gel Electrophoresis System is compatible for use with Components multichannel pipettors or automated liquid handling systems The system consists of the following components E Gel 96 gels see below and next page Mother E Base and Daughter E Base page 16 E Holder Platform page 17 E Editor 2 02 Software page 17 Applications E Gel 96 agarose gels are suitable for analyzing multiple samples 14 PCR products Restriction digests RT PCR reactions Library screenings SNPs analysis Continued on next page E Gel 96 Agarose Gels E Gel 96 Gels E Gel 96 gels are self contained pre cast agarose gels that include agarose a proprietary buffer system ethidium bromide or SYBR Safe DNA stain and electrodes packaged inside a dry disposable UV transparent cassette Each E Gel 96 gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format see figure on the next page The wells of the E Gel 96 gel are compatible with the standard 96 well plate format for automated loading See page 27 for product specifications In addition each E Gel 96 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial barcode readers page 98 The lane numbers are labeled with fluorescent dye that transfers to the image a
13. heat and denaturing agent Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal No IR coating on camera when using an UV system Used a UV light source to visualize DNA Solution Wait 10 15 minutes for gel to cool before visualization Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button or Go button for 2 seconds to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page 123 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel EX agarose gels empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use a blue light transilluminator such as the Safe Imager Blue Light Transilluminator Invitrogen Cat no 537102 69 DNA Purification Using E Gel CloneWell Agarose Gels Sample Preparation for E Gel CloneWell Agarose Gels Introduction Materials Needed Amount of DNA Total Sample Volume Preparing Samples Loading Buffer Loading Method 70 E Gel CloneWell pre cast 0 8 agarose
14. is a registered trademark of Microsoft Corporation 133 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
15. 200 ng 250 bp DNA Ladder 400 ng pecDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng 1 Kb Plus DNA Ladder 300 ng 1 Kb Plus DNA Ladder 300 ng 1 kb PCR fragment 2 kb PCR fragment 3 kb PCR fragment 250 bp DNA Ladder 400 ng High DNA Mass Ladder 200 ng Continued on next page Results with E Gel Single Comb Gels Continued 2 Single Comb Results obtained using a 2 agarose gel are shown below using 20 ul per lane Gel Digestion of pUC18 and pcDNA 3 1 are as described on the previous page Lane Sample Low DNA Mass Ladder 250 ng 100 bp DNA Ladder 300 ng pcDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng 50 bp DNA Ladder 300 ng 50 bp DNA Ladder 300 ng 450 bp PCR fragment 500 bp PCR fragment 10 1kbPCR fragment 11 100bp DNA Ladder 300 ng 12 Low DNA Mass Ladder 250 ng 2 Agarose GP 3 4 5 6 fF B 8 WW ii 12 O CON D OFF amp W N 4 Single Comb Results obtained using a 4 agarose gel are shown below using 20 ul per lane Gel Lane Sample 50 bp DNA Ladder 300 ng 50 bp DNA Ladder 300 ng 450 bp PCR fragment 500 bp PCR fragment 25 bp DNA Ladder 400 ng 25 bp DNA Ladder 400 ng 10 bp DNA Ladder 740 ng 10 bp DNA Ladder 740 ng 450 bp PCR fragment 10 500 bp PCR fragment 11 50bp DNA Ladder 300 ng 12 50bp DNA Ladder 300 ng 4 Agarose HA 12 345S F7 8 8 10 11 12 Oo COND OFF WN FR Continued on next page 41 Results
16. 96 gel note that the barcode label is easily overexposed To ensure that the barcode label is distinct and readable in the image experiment with different shutter settings for your particular camera Loading E Gel 48 and 96 Gels Continued Aligning the The wells of the E Gel 96 gel are staggered to provide maximum run length see Robotic Loading Figure 1 below For proper loading of samples it is important to program your Assembly robotic loading system to set the A1 tip of the 8 12 or 96 tip robotic head over the E Gel 96 gel cassette as described below Set the position of the first tip approximately 1 mm above the slope of the A1 well see Figure 2 below This will ensure that the remaining tips are aligned above the slopes of the remaining wells Refer to the manufacturer s manual of your robot to program this setting After programming the setting load your samples During loading the samples will fall onto the slopes of the wells and be drawn into the wells by capillary force Figure 1 4 5 6 7 8 9 1011 12 M Cayce LN L3 L3 L3 aad CHO Io fH Hf nr COC GO ii Lr A B C CH LJ 3 L3 aa aa CHC aa CAO D LL pr m pmr O C O O DOC HH AO EJ FOI 3 q Uo O3 DO Oooo oo F L E E Ej E COO EE O O O C G CH L3 L3 L3 3 L3 L3 L3 L3 CECH CAO H FLDprrm prm CO O DO Hf CO LT Figure 2 Continued on next page 99 Loading E Gel 48 and 96 Gels Continued Important Selecting Program on E Base gt
17. Electrophoresis bases E Gel pre cast agarose gels are self contained gels that include electrodes packaged inside a dry disposable UV transparent cassette The E Gel agarose gels run in a specially designed device that is a base and power supply combined into one device two bases are available for running E Gels the new iBase system and the original economical E Gel Powerbase Using E Gel agarose gels for electrophoresis of DNA samples offer the following advantages e Provides fast safe consistent high resolution electrophoresis e Eliminates the need to prepare agarose gels and buffers and to stain gels e Compatible with most commercially available robotic systems for high throughput agarose gel electrophoresis e Available in a variety of agarose percentages well formats and throughput capacities to suit your applications e Offered with a number of different DNA gel stains to accommodate your application e Includes E Gel CloneWell and E Gel SizeSelect gels to accelerate and simplify DNA gel purification and improve cloning results Three categories of E Gel agarose electrophoresis systems are available from Invitrogen based on your throughput requirements e Low Throughput E Gel Electrophoresis System designed for electrophoresis of 8 16 DNA samples per gel e Medium Throughput E Gel Electrophoresis System designed for electrophoresis of 48 DNA samples per gel This system is compatible for use w
18. Ladder 12373 031 markers in a volume of 20 ul in marker well Use a buffer containing the same salt concentration as your samples Continued on next page 97 Loading E Gel 48 and 96 Gels Introduction Note Using the Barcode 98 This section describes the procedure for loading and running samples on E Gel 48 and E Gel 96 gels using the Mother E Base and Daughter E Base The Mother E Base and Daughter E Base are designed to fit most robotic platforms allowing you to load and run E Gel 48 and 96 Gels directly on the robot If you need to load multiple gels on a robotic platform while other gels are running on the E Base use an E Holder Platform page 107 for details e The E Gel 48 and 96 gels can only be used once Do not re use e The E Gel 48 and 96 gels are compatible with the E Gel 96 mother base and daughter base available previously from Invitrogen For instructions on using the gels with E Gel 96 mother base and daughter base see page 118 Each E Gel 48 and 96 gel is labeled with an individual barcode with a number The barcode facilitates identification of each gel cassette during the electrophoresis of multiple gels Each E Gel 48 and 96 gel contains an EAN 39 type of barcode which is recognized by the majority of commercially available robotic barcode readers Refer to the manufacturer s instructions to set up the barcode reader Note When capturing an image of the E Gel 48 or
19. O Box 4035 Ness Ziona Israel 74103 N The Caution symbol denotes a risk of safety hazard Refer to accompanying Caution documentation The E Gel Safe Imager Real time Transilluminator is classified as a Class 1 LED 5 product which is indicated by the symbol to the left A yellow label is affixed to the side of the E Gel Safe Imager Amber filter saying Caution Class 2 LED radiation when open do not stare into the beam 130 Technical Support Web Resources Visit the Invitrogen Website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Fax 44 0 141 814 6117 tech_su
20. a blue light transilluminator such as the Safe Imager Blue Light Transilluminator Invitrogen Cat no 37102 Use the REVERSE program of the iBase Power System to run the band backwards into the collection well see the iBase Power System manual Refill the second row with sterile water until the well is full prior to running your band of interest into the collection well Collect DNA from the well in two or more fractions Be sure to load the recommended DNA amount DNA Purification Using E Gel SizeSelect Agarose Gels Sample Preparation for E Gel SizeSelect Agarose Gels Introduction Materials Needed Amount of DNA Total Sample Volume Preparing Samples E Gel SizeSelect 2 pre cast agarose gels feature two rows of wells a top row for loading samples and a bottom row to retrieve your DNA bands of interest The gels contain a proprietary fluorescent nucleic acid stain that is visualized by blue light transilluminator excitation emission at 490 522 nm and allows detection down to 1 5 ng band of DNA The recovered DNA has been shown to be compatible with nick translation and amplification steps without further purification For optimal results follow the guidelines for preparing your DNA sample described in this section e DNA sample e Loading buffer with diluted tracking dye optional e Molecular weight markers page 57 For optimal results refer to the following tables when determining the amou
21. agarose gels using the E Gel iBase Power System Place the amber filter over the E Gel iBase Toggle between program minutes and seconds on the iBase by pressing the Mode button until the program blinks Select the program Run SizeSelect 2 program 8 using the Up Down A V buttons to change the program Note The iBase Power System has the option of speed runs if you want quick results See below for more information p in B35 Set time to Run Time to Reference Line as listed in the Run Time Estimation Table for the appropriate band size Default run time for E Gel SizeSelect is 8 minutes If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons up to the maximal run time of 20 minutes Reference Line Reference Line f 4 VT Tl Bands reaching the Reference Line Continued on next page 89 Running E Gel SizeSelect Agarose Gels Continued Electrophoresis Using iBase Power System continued 90 The end of the run is signaled with a flashing red light and rapid beeping If the band has not reached the reference line run the gel longer until the band reaches the line Press and release the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program When the band reaches the reference line refill the collection wells
22. and Safe Imager Blue Light Transilluminator Invitrogen Cat nos G6500 and S37102 are compatible for use with E Gel EX agarose gels Refer to the next section for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager Blue Light Transilluminator See page 22 for details Blue light transilluminators available from other manufacturers are also compatible for use with E Gel EX agarose gels e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel EX agarose gels using a CCD camera or a laser based scanner For photographing gels refer to page 125 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel EX agarose gels E Gel EX agarose gels have greater sensitivity than ethidium bromide stained gels As a result a shorter exposure time or lower gain setting may be necessary 63 Opening an E Gel EX Agarose Gel Cassette Introduction The E Gel EX agarose gel has a novel openable format that allows the cas
23. cloning efficiencies compared to UV transillumination Safe Imager Blue Light E Gel Safe Imager Transilluminator Real time Transilluminator Viewing surface 20 x 20 cm 6 2 x 7 7 cm dimensions Overall dimensions 28 x 31 x 7 cm 20 0 x 11 0 x 4 3 cm Lamp life 50 000 hours 50 000 hours Included accessories Amber filter unit and Amber filter unit and viewing glasses for viewing glasses for viewing results viewing results Emission maxima 470 nm 480 nm Make sure to use the E Gel Safe Imager Amber filter unit or E Gel Safe Imager viewing glasses they help to visualize the SYBR Safe stained DNA and also prevent prolonged exposure of your eyes to the intense blue light The Safe Imager Transilluminators are designed for viewing stained gels on the Emission spectra for the Safe Imager Transilluminator laboratory bench top E Safe Imager Uu transilluminators are compatible with E Gel E with SYBR Safe gels E Gel EX gels E Gel pp ud i e 350 400 450 500 550 600 Gel SizeSelect gels Wavelen eth nm Light from a LED source within the Safe Imager Blue Light Transilluminator passes through a blue filter producing a single intensity signal at approximately 470 nm see above effective for the excitation of SYBR DNA binding dyes such as SYBR Safe DNA gel stain as well as many of our protein gel stains such as SYPRO Ruby SYPRO Orange and Pro Q Diamond stains Sensitivity obtained
24. connected to a Mother E Base Once connected to a Mother E Base each Daughter E Base is designed to function independently of the Mother E Base or other Daughter E Bases Mother E Base Daughter E Base Mother 7 E Base Daughter E Base The E Holder Platform is designed to hold E Gel 96 gels during robotic loading Use the E Holder when you need to load multiple gels on a robotic platform while the other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run gels The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and documentation Capture an image of the gel and then use the E Editor 2 02 software to e Align and arrange the lanes in the image e Save the reconfigured image for further analysis e Copy and paste selected lanes or the entire image into other applications for printing saving e mailing and or publishing on the Web The E Editor 2 02 software can be downloaded for free at http www invitrogen com egels and following the instructions to download the software and user manual 17 Nucleic Acid Gel Stains for E Gel Agarose Gels Available Nucleic Acid Gel Stains Advantages of SYBR Safe DNA Gel Stain Features of Proprietary Fluorescent Nucleic Acid Gel Stain 18 E Gel agarose gels come in
25. connected to a mains socket outlet with protective earthing connections e Ventilation requirements no special requirements The E Gel iBase Power System complies with part 15 of the FCC rules Operation of the device is subject to the following two conditions e The device may not cause harmful interference e The device must accept any interference received including interference that may cause undesired operation Ethrog Biotechnologies Ltd an Invitrogen company is the manufacturer and owner of the UL file For more information contact Ethrog Biotechnologies Ltd Ness Ziona Science Park Bldg 22 P O Box 4035 Ness Ziona Israel 74103 For more information contact Technical Support see page 131 E Gel The E Gel PowerBase complies with the Underwriters Laboratories Inc PowerBase regulation and is listed under file no E189045 in the US and Canada This device complies with part 15 of the FCC Rules Operation is subject to the following two conditions e This device may not cause harmful interference UL e This device must accept any interference received including interference that CN E US may cause undesired operation Continued on next page 129 Explanation of Symbols and Warnings Continued E Gel iBase The E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator comply with the Underwriters Laboratories Inc regulation and the European Community Safety requirements Opera
26. downloaded for free from our Website at www invitrogen com egels System The Medium Throughput E Gel Electrophoresis System is compatible for use Components with multichannel pipettors or automated liquid handling systems The system consists of the following components e E Gel 48 gels see below and next page e Mother E Base and Daughter E Base page 16 e E Editor 2 02 Software page 17 Applications E Gel 48 agarose gels are suitable for analyzing multiple samples e PCR products e Restriction digests e RT PCR reactions e Primer dimers 4 E Gel 48 gels e Library screenings e Diced dsRNA 4 E Gel 48 gels e SNPs analysis Continued on next page 12 E Gel 48 Agarose Gels E Gel 48 Gels Separation Range for E Gel 48 Gels E Gel 48 gels are self contained pre cast agarose gels that include agarose a proprietary buffer system ethidium bromide and electrodes packaged inside a dry disposable UV transparent cassette Each E Gel 48 gel contains 48 sample lanes and 4 marker lanes and is designed for medium throughput agarose electrophoresis of nucleic acids This configuration provides a 3 2 cm run length See page 27 for product specifications The 4 6 E Gel 48 gels are prepared with high resolution agarose to ensure quality resolution of DNA fragments below 400 bp see next page for separation range The wells of the E Gel 48 gel are compatible for loading with a multichannel pipettor The lane
27. four different formats for staining your DNA e Regular E Gel agarose gels contain the standard DNA gel stain ethidium bromide e E Gel with SYBR Safe contains SYBR Safe DNA gel stain which is not classified as hazardous waste under US Federal regulations and improves cloning efficiency when using blue light for imaging e E Gel EX and E Gel SizeSelect agarose gels contain a proprietary fluorescent nucleic acid stain compatible with blue light visualization for increased nucleic acid detection sensitivity SYBR Safe DNA gel stain is a safer more environmentally friendly alternative to ethidium bromide and offers the following advantages e SYBR Safe DNA gel stain is not classified as hazardous waste under US Federal regulations and meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations e SYBR Safe DNA gel stain does not cause mutations chromosomal aberrations or transformations in appropriate mammalian test systems in contrast to ethidium bromide which is a strong mutagen e Asingle oral administration of SYBR Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5000 mg kg e Visualizing E Gel with SYBR Safe using blue light transilluminators dramatically reduces DNA damage that lowers cloning efficiency For details on SYBR Safe DNA gel stain see page 18 The proprietary fluorescent nucleic acid stain in E Gel EX and E G
28. migrate between adjacent wells in the row below For example the bands of lane A2 will migrate between wells B1 and B2 The box highlights a lane The gel contains the following samples Lane 1233 4 5 6 10 11 12 7 8 9 M Sample 1 kb PCR product 100 ng 3 kb PCR product 100 ng 9 kb PCR product 100 ng E Gel High Range DNA Marker 113 Results with E Gel 96 Gels Continued 2 E Gel 96 Gel 114 Results obtained using a 2 E Gel 96 gel are shown in the figure below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS120 system You can use a mini transilluminator to view the bands You can vary the amount of markers loaded to improve gel imaging NELLE Lu Leu 4 4 4 The gel contains the following samples Lane Sample 1 2 3 10 11 12 125 bp PCR product 100 ng 4 5 6 240 bp PCR product 100 ng 7 8 9 1 kb PCR product 100 ng M E Gel Low Range Quantitative DNA Ladder Using E Editor 2 02 Software Introduction Imaging the Gel Downloading Software The E Editor 2 02 software for Windows allows you to reconfigure digital images of E Gel 48 and E Gel 96 gels for analysis and documentation The staggered lanes in an E Gel 96 gels are difficult to compare and analyze by standard 1 D gel analysis programs such as Bio Rad s Quantity One Phoretix 1D or Kodak 1D software E Editor 2 02 software r
29. per lane for samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution The recommended total sample volume for each gel type is listed in the table below Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Gel Type Total Sample Volume E Gel single comb gel 20 ul E Gel double comb gel 20 ul Prepare your samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 ul For samples that are in a high salt buffer refer to page 31 Loading buffer is optional See page 30 for more details Load samples in the appropriate sample volume directly into the wells The E Gel agarose gel should be loaded within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples Continued on next page Sample Preparation for E Gel continued DNA Molecular Weight Markers We recommend using the following DNA molecular weight markers for different types of E Gel agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel agarose gels containing ethidium bromide Single comb E Gel gels E Gel 1 Kb Plu
30. properly grounded AC outlets and cords Installing iBase If using the iBase Power System and Safe Imager Real time Transilluminator and Safelmager 1 Place the iBase directly onto the E Gel Safe Imager Real time Transilluminator so that the legs of the iBase fit directly into the grooves of the Safe Imager as shown in the image below 2 Plug the short electrical cord of the E Gel Safe Imager Real time Transilluminator a into the power inlet of the iBase b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager c and connect the power cord to the electrical socket To power outlet Continued on next page 86 Loading E Gel SizeSelect Agarose Gels Continued Inserting the 4 E Gel SizeSelect Agarose Gel Important Do not pre run E Gel SizeSelect agarose gels Open the package and remove the gel Gently remove the combs from the upper and lower wells of the E Gel SizeSelect agarose gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Slide the cassette into the two electrode connections on the E Gel iBase Power System Press firmly at the left edge of the cassette to secure it into the iBase The two electrodes on the right side of the gel cassette must be in contact with
31. single beep every minute The LCD displays Run Complete Press Go 6 Press and release the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program 7 Remove the E Gel cassette from the iBase You are now ready to proceed to imaging or any other application with the gel Speed Runs The iBASE is pre programmed with a program for quick runs to get a Using iBase yes no result The program SPEED E Gel utilizes high power and is suitable for 1 2 and 2 E Gels with SYBR Safe This program is limited to 7 minutes where the bands migrate less than half the length of the gel A run exceeding 7 minutes under these conditions results in a defective run Continued on next page 49 Running E Gel with SYBR Safe Gels Continued Interrupting a Run Using iBase You can interrupt an electrophoresis run on the iBase at any time by pressing and releasing the Go button to stop the current The stopped current is indicated by a flashing red light and the digital display flashes to indicate that the run was interrupted The display also shows Press GO to Run Hold Go to Reset You can remove the gel from the iBase to check the progress of the run Then Electrophoresis 1 Using PowerBase v 4 To continue the run from the point at which it was stopped reinsert the gel and press and release the Go button The light changes to steady green and the LC
32. system for E Gel with optimal SYBR Safe empirically You may need to increase the exposure time or gain setting Stripes visible on No IR coating on camera Use IR blocking filter or emission filter with IR image SYBR when using an UV system coating Safe gel 117 Appendix Using E Gel 96 Mother Daughter Base Introduction Using E Gel 96 Mother Base and Daughter Base 118 Instructions are provided below to perform electrophoresis of E Gel 48 and 96 gels with E Gel 96 mother base and daughter base previously available from Invitrogen For instructions on using the Mother E Base and Daughter E Base see page 103 Note The E Gel 96 mother base and daughter base are designed differently than Mother and Daughter E Base The recommended run time for E Gel 48 gels is 20 minutes and E Gel 96 gels is 12 minutes 1 Connect the electrical plug from the E Gel 96 mother base to an appropriate electrical outlet 110 V or 220 V If a gel cassette is not inserted the light on the mother base is not illuminated If you are using an E Gel 96 daughter base connect the daughter base to a mother base A red light at the lower left corner of the mother base and daughter base will illuminate when the E Gel 48 or 96 cassette is correctly inserted and the digital display will show the default time e g 12 minutes or the last programmed time To begin electrophoresis press and release the power butt
33. the gel followed by a second round of loading into the remaining wells or robotic loading devices 8 or 12 tip E Gel 96 Manual multichannel pipettor or robotic loading devices 8 12 or 96 tip One Step Loading Load DNA samples into the gel as described below see page 96 for sample Method preparation Avoid introducing bubbles while loading as they will cause the bands to distort The gel should be loaded within 15 minutes of removal from its plastic pouch Load prepared samples into each well e For E Gel 48 Gels Load 15 ul of prepared sample into sample wells Load 15 ul of sample buffer containing the same salt concentration as your sample into any remaining empty wells e For E Gel 96 Gels Load 20 ul of sample into each well Load 20 ul of sample buffer containing the same salt concentration as your sample into any remaining empty wells Load the appropriate DNA markers page 97 in the marker M wells of an E Gel 48 and 96 gels 102 Running E Gel 48 and 96 Gels Using E Base Instructions for running E Gel 48 and E Gel 96 gels in a Mother E Base or Daughter E Base are provided below Note It is not necessary to have a gel in the Mother E Base if you are using a Daughter E Base However the Mother E Base must be plugged into an electrical outlet 1 To begin electrophoresis press and release the pwr prg button located on the lower right corner of the Mother E Base The re
34. to 25 ul with sterile water The refill volume may vary between wells Do not overfill Enter the appropriate time listed under Run Time from Reference Line to Collection Well from the Run Time Estimation Table for your band Press Go to run the gel Monitor the run carefully As the run ends the band of interest may be seen migrating into the collection well Bands that have entered the Collection Well Collect DNA from the wells using a pipette without piercing the bottom of the well Proceed to your application using the collected DNA If the band of interest has overshot the collection well use the Reverse E Gel program to run the band back into the collection well Refer to the E Gel Technical Guide for instructions Remove the E Gel SizeSelect cassette from the iBase You are now ready to proceed to imaging or any other application with the gel Additional DNA bands can be collected from the same well s Be sure to refill the collection wells with more water as water is lost during the run Continued on next page Running E Gel SizeSelect Agarose Gels Continued Interrupting a Run You can interrupt an electrophoresis run on the iBase at any time by pressing Using iBase and releasing the Go button to stop the current The stopped current is indicated by a flashing red light and the digital display flashes to indicate that the run was interrupted The display also shows Press GO to Run Hold Go to Re
35. transilluminator with abrasive cleaners sharp instruments or harsh solvents Before cleaning the instrument disconnect it from the electrical outlet Product Specifications E Gel Single Comb and Double Comb Gel Specifications E Gel 48 Gel Specifications E Gel 96 Gel Specifications The E Gel cassette is 8 cm x 10 cm and 0 6 cm thick The thickness of the E Gel gel is 3 mm and the volume of the gel is 20 ml e Single comb gel Each well is 4 1 mm wide and the space between wells is 1 mm The running distance is 5 8 cm Each gel contains 12 lanes e Double comb gel The sample well is 4 6 mm wide and the marker well is 2 8 mm wide The running distance from each comb is 2 9 cm Each gel contains two rows of 8 sample wells and 2 marker wells M The wells of the double comb gel are compatible for loading with a multichannel pipettor Each E Gel 48 gel contains 48 sample wells and 4 marker wells M Cassette Size 13 5 cm 1 x 10 8 cm w x 0 67 cm thick Gel Thickness 3 7 mm Gel Volume 50 ml Gel Percentage 196 296 and 496 Well Depth 3mm Dimensions of the Well 3 6 mm 1 x 2 2 mm w Running Distance 32 mm one well to the next Space between Well Center 4 5 mm The wells of the E Gel 48 gel are compatible for loading with a multichannel pipettor Each E Gel 96 gel contains 96 sample wells and 8 marker wells M Cassette Size 13 5 cm 1 x 10 8 cm w x 0 67 cm thick Gel Thickness 3 7 mm
36. using this instrument is comparable to that obtained with a standard UV transilluminator Continued on next page Safe Imager Blue Light Transilluminators Continued E Gel Safe Imager Real time Transilluminator Light Source LED Indicator light 4 ON OFF button USB port Power inlet Attached short electrical cord E Gel Safe Imager Real time Transilluminator The E Gel Safe Imager Real time Transilluminator is designed for viewing E Gel with SYBR Safe E Gel CloneWell E Gel EX and E Gel SizeSelect gels on the laboratory bench top for real time monitoring on the E Gel iBase Power System or for documentation purposes at the end of the run directly on the E Gel Safe Imager E Gel Safe Imager Real time The E Gel Safe Imager Real time Transilluminator top Transilluminator has the following features e Anarray of 12 LED sources behind a blue filter that emit high intensity blue light e A red ON OFF button located at the front e 30 seconds and 5 minutes automatic shut off options e ALED indicator light just behind the ON OFF button to indicate the status of the E Gel Safe Imager Real time Safe Imager Transilluminator back e A short electrical cord to connect to the iBase e USB port to enable future program updates Light from the array of 12 Emission spectrum for the E Gel Safe Imager LED sources within the Real time Transilluminator
37. wells See table on page 126 for volume to load in the first step Do not premix with sample Load 10 pl of sample with loading buffer per sample well Load 10 pl of the appropriate molecular weight markers with loading buffer into the marker well page 57 4 Load 10 ul of water loading buffer may be added into any remaining empty wells Dilute samples with glycerol loading buffer next page glycerol in deionized water or glycerol in TE buffer to obtain a final glycerol concentration of 10 in a final sample volume of 10 ul see page 31 for details 127 Explanation of Symbols and Warnings E Base The Mother E Base and Daughter E Base comply with the Underwriters Laboratories Inc regulation and the European Community Safety requirements Operation of the E Gel bases is subject to the following conditions e Indoor use e Altitude below 2 000 meters UL us e Temperature range 5 to 40 C LISTED e Maximum relative humidity 80 Eas e Installation categories over voltage categories II Pollution degree 2 e Mains supply voltage fluctuations not to exceed 10 of the nominal voltage 100 240V 50 60Hz 1500 mA e The Mother E Base has been tested with up to 3 Daughter E Bases connected at one time e Mains plug is a disconnect device and must be easily accessible e Do not attempt to open E Base devices To honor the warranty E Base can only be opened and serviced by Invitrogen e The protection pro
38. will show the count down time while the run is in progress 5 TheiBase will signal the end of the run with a flashing red light and rapid beeping for 30 seconds followed by a single beep every minute while the LCD display will read Run Complete Press GO 6 Press and release the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program 7 Remove the E Gel cassette from the iBase You are now ready to proceed to imaging or any other application with the gel e E Gel SizeSelect agarose gels can only be used once Do not re use them Note e E Gel SizeSelect agarose gels already contain a proprietary DNA gel stain and therefore do not have to be stained after the electrophoresis run Continued on next page 91 Visualizing E Gel SizeSelect Agarose Gels Introduction The proprietary DNA gel stain in E Gel SizeSelect agarose gels has an excitation maxima at 490 nm and an emission maximum at 522 nm when bound to nucleic acid Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel see below for details Viewing E Gel View E Gel SizeSelect agarose using these instruments SizeSelect e Blue light transilluminator The E Gel Safe Imager Real time Trans Agarose Gels illuminator and Safe Imager Blue Light Transilluminator Invitrogen Cat nos G6500 and 37102 are compatible for us
39. with E Gel Double Comb Gels Introduction 0 8 Double Comb Gel 2 Double Comb Gel Note 42 Results obtained using double comb E Gel gels are shown below All gels were photographed using a Kodak EDAS120 system You can also use a mini transilluminator to view the bands Note You may vary the amount of markers loaded to improve photography of the gel Results obtained using a 0 8 double comb E Gel gel are shown below 10 ul loaded in M lanes 20 ul loaded in sample lanes Digestion of pUC18 and pcDNA 3 1 are as described for the 0 8 single comb gel page 40 Lane Sample 1 High DNA Mass Ladder 200 ng 2 pcDNA3 1 Nco I cut 150 ng 3 pcDNA3 1 uncut 120 ng 4 pUC18 Pst I 60 ng M Low DNA Mass Ladder 125 ng 5 1kb PCR fragment 6 3kb PCR fragment 7 5kb PCR fragment 8 High DNA Mass Ladder 200 ng TIE IE Tt a ee ey Lanes 9 16 contain samples as described for Lanes 1 8 Results obtained using a 2 double comb E Gel gel are shown below 10 pl loaded in M lanes 20 ul loaded in sample lanes Digestion of pUC18 and pcDNA 3 1 are as described for the 0 8 single comb gel page 40 Lane Sample 1 Kb Plus DNA Ladder 300 ng pcDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng Low DNA Mass Ladder 125 ng 500 kb PCR fragment 1 kb PCR fragment 2 kb PCR fragment 1 Kb Plus DNA Ladder 300 ng Lanes 9 16 contain samples as described for Lanes 1 8 iP Doublecomb A
40. 0 bp 2 kb 400 bp 10 kb 50 bp 3 kb 10 bp 400 bp 1 kb 10 kb 100 bp 2 kb The table below lists the power systems compatible with the various types of E Gel agarose gels E Gel iBase E Gel Power System PowerBase v 4 b N Y Y Y N Y N Y Y N N Mother and Daughter E Base Integrated Power Supply Z Z F N Y The E Gel iBase Power System is compatible with the E Gel Safe Imager Real time Transilluminator The E Gel PowerBase v 4 is compatible with the Safe Imager blue light transilluminator Continued on next page Gel Selection Continued Advantages of E Gel EX Agarose Gels Advantages of E Gel with SYBR Safe Agarose Gels Advantages of E Gel CloneWell Agarose Gels Advantages of E Gel SizeSelect Agarose Gels E Gel EX pre cast agarose gels are general use gels which contain a proprietary fluorescent nucleic acid stain with high sensitivity allowing e Detection down to 1 ng band of DNA e Compatibility with blue light transillumination to dramatically reduce DNA damage e Separation of RNA e Easy opening of cassette with gel knife E Gel with SYBR Safe contains SYBR Safe DNA gel stain instead of ethidium bromide Use E Gel with SYBR Safe to e Minimize your hazardous waste since SYBR Safe DNA gel stain is not classified as such under US Federal regulations e Protect yourself and your co workers because E Gel with SYBR Safe eliminates
41. 103 Running E Gel 48 and 96 Gels Continued Note Interrupting an Electrophoresis Run Maintaining E Base 104 We recommend that you disconnect the Mother E Base from the electrical outlet when not in use for a prolonged period of time You can interrupt an electrophoresis run at any time by pressing and releasing the pwr prg button to stop the current The stopped current is indicated by a steady red light and the digital display will flash to indicate that the run was interrupted You can remove the gel from the E Base to check the progress of the run Then e Tocontinue the run from the point at which it was stopped reinsert the gel and press and release the pwr prg button The light changes to steady green and the digital display shows the count down time e To cancel the rest of the interrupted run press and hold the pwr prg button for a few seconds The digital display will reset and the base will return to Ready Mode If desired you can then program a new run time as described on page 101 and rerun the gel In case of an external power failure loss of electricity or the electrical cord is accidentally removed from the outlet the run will continue when the power resumes The Mother E Base or Daughter E Base will signal the end of the run as described on the previous page except the light will be an alternating red green to indicate that an external power failure had occurred during the run The surfaces o
42. 4 Time 17 minutes The default time is 12 minutes Change the time manually by pressing and holding the time button until the run time appropriate for your type of gel is displayed Continued on next page Loading E Gel 48 and 96 Gels Continued Setting the Time Inserting Gel in the E Base The initial default time setting on an E Base for program EG is 12 minutes Follow instructions below to increase or decrease the time setting if desired Do not run an E Gel 96 gel for more than 20 minutes or E Gel 48 gel for more than 30 minutes To increase or decrease the default run time when no cassette is inserted on the base use the following steps 1 Connect the Mother E Base to an electrical outlet If you are using a Daughter E Base connect the Daughter E Base to the Mother E Base and then connect the Mother E Base to an electrical outlet 2 Press and release the time button located on the lower right corner of the base to view the time setting 3 Press and hold the time button to increase the time continuously When you reach the desired default time release the time button If the time button is not released the time setting will increase until it reaches 00 To begin cycling through the numbers again starting from 00 press the time button again Note To increase the run time when a cassette is inserted press and release the time button to increase the time setting by 1 minute intervals or press and ho
43. 5 2 pe ND Ope em ge emque dos We have adjusted the brightness and contrast to improve the reproduction quality of the E Gel gel images in this manual Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel single comb and double comb gels No current Copper contacts in the Make sure the copper contacts in the base are intact base are damaged Expired or defective Use fresh gel cassette Use properly stored gels before the gel cassette specified expiration date E Gel cassette isnot Remove cassette and reinsert a steady red light inserted properly into a illuminates on the base when the cassette is correctly base inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase and PowerBase Poor resolution or Sample is overloaded Load 20 100 ng of sample DNA per band Less DNA is smearing of bands required since E Gel agarose gels are thinner High salt concentration Dilute your high salt samples as described on page 31 Very low volume of Avoid introducing bubbles while loading the samples sample loaded or Bubbles will cause band distortion sample was not loaded Load the recommended sample volume based on the gel properly type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any
44. 5 C 5 minutes Total RNA 65 C 5 minutes Total RNA formamide 65 C 5 minutes Total RNA formamide 65 C 5 minutes RNA Ladder native heat Ladder formamide 65 C 5 minutes SEE BI Ead 1 tween i tprp nn Ner i EX Agarose Gel invitrogen b T e n sc Continued on next page 67 Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel EX agarose gels No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before cassette the specified expiration date E Gel EX cassette is not Remove cassette and reinsert a steady red light inserted properly into a base illuminates on the base when the cassette is correctly inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Poor resolution Sample is overloaded Do not load more than the recommended amount of or smearing of DNA sample per band see page 56 bands High salt concentration Dilute your high salt samples as described on page 31 Aberrant pre run step Do not pre run E Gel EX agarose gels Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properl
45. 9 for details Safe Cassette Disposal of E Gel SYBR Safe DNA gel stain shows no or very low mutagenic activity when tested with SYBR Safe by an independent licensed testing laboratory and this stain is not classified as hazardous waste under US Federal regulations As disposal regulations vary please contact your safety office or local municipality for appropriate SYBR Safe disposal in your community 50 Visualizing E Gel with SYBR Safe Agarose Gels Introduction Viewing E Gel with SYBR Safe Imaging E Gel with SYBR Safe Required Filters Important Exposure Time and Gain Setting Bound to nucleic acids SYBR Safe DNA gel stain has fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate see below View E Gel with SYBR Safe using these instruments e Blue light transilluminator The E Gel Safe Imager Real time Trans illuminator and Safe Imager Blue Light Transilluminator Invitrogen Cat nos G6500 and 37102 are designed specifically for use with SYBR Safe stained DNA gels Refer to page 22 for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager Blue Light Transilluminator Blue light transilluminators available from other manufacturers are also compati
46. 91 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com Continued on next page Purchaser Notification Continued Limited Use Label License No 223 Labeling and Detection Technology Limited Use Label License No 188 SYBR Safe Nucleic Acid Stain The manufacture use sale or import of this product may be subject to one or more pending US patent applications and corresponding foreign equivalents owned by Invitrogen Corp The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for pro t entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party f
47. Agarose Gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette 2 Load 20 25 pl prepared sample per sample well in the top row see page 70 for sample preparation 3 Load 5 10 ul of the appropriate molecular weight marker page 71 in the small middle well Load 25 ul of water into any remaining empty wells of the top row Load 25 30 ul of water into the wells of the bottom row Top row 4 to load Sample Small middle well for Molecular Weight Markers Bottom row to retrieve DNA E Gel win SYBR Safe 4 A Load Samples and Molecular Weight Marker in the top row and water in the bottom row Running E Gel CloneWell and Collecting DNA Introduction After you have loaded your samples you are ready to proceed with electrophoresis and retrieve your DNA This consists of three steps 1 Run your fragments to reach the reference line just above the bottom row 2 Runthe fragments from the reference line into the bottom well while monitoring the progress constantly 3 Retrieve your fragment from the bottom well Instructions are provided below to run an E Gel CloneWell agarose gel using the E Gel iBase Power System Monitoring the The progress of the E Gel CloneWell agarose gel needs to be monitored E Gel CloneWell during the run with a blue ligh
48. CloneWell 0 8 60 5 E Gel CloneWell Reverse run REVERSE E Gel 6 E Gel 0 8 1 2 2 SPEED E Gel 7 E Gel EX 1 2 E Gel EX 20 8 E Gel SizeSelect 2 SizeSelect 2 20 This mode is not compatible with E Gel 4 E Gel EX E Gel CloneWell or E Gel SizeSelect gels 124 Filter Selection Guide Filter Selection Use the filter recommended with your instrument below to photograph E Gel Guide with SYBR Safe E Gel EX or E Gel SizeSelect agarose gels We have shown the most popular instruments other instruments with an excitation source in the UV range or between 470 530 nm may also be used with the proper filter Contact your instrument manufacturer for advice Instrument Manufacturer Excitation Source Emission Filter Alphalmager Alpha Innotech 302 nm SY B 500 Alphalmager HP Alpha Innotech 302 nm SYB 500 AlphaDigiDoc RT Alpha Innotech UV transilluminator Shroud Camera Stand Alpha Innotech UV transilluminator SYB 100 DE500 or DE400 light cabinet 2 17 diameter UV transilluminator SYB 500 Alpha Innotech DE500 or DE400 light cabinet 2 diameter UV transilluminator SYB 400 Alpha Innotech VersaDoc Imaging Systems Bio Rad Broadband UV 520LP Molecular Imager FX Systems Bio Rad 488 nm 530 nm BP Gel Doc Systems Bio Rad 302 nm 520DF30 170 8074 Typhoon 9400 9410 GE Healthcare 488 nm 520 BP 40 Typ
49. D display shows the count down time It is also possible to change the remaining run time but not the program as described on the previous page before continuing the run To cancel the rest of the interrupted run press and hold the Go button for a few seconds The LCD display will reset and the base will return to Ready Mode If desired you can then select a new program or run time as described on the previous page and rerun the gel Choose the 30 minute run on the PowerBase v 4 for E Gel with SYBR Safe gels Press and release the 30 min button to start the electrophoresis run The light will change to a steady green light Note The actual running time may vary between 30 33 minutes 2 Current through the E Gel gel automatically shuts off at the end of each run The E Gel PowerBase v 4 signals the end of the run with a flashing red light and rapid beeping 3 Press and release either button to stop the beeping The light will turn to a steady red light 4 Atthe end of the run remove the gel cassette from the power unit and analyze your results as described in the next sections e E Gel agarose gels can only be used once Do not re use them Note e E Gel with SYBR Safe gels already contain the SYBR Safe DNA gel stain and therefore do not have to be stained after the electrophoresis run Opening the To open the E Gel with SYBR Safe cassette for excision of DNA fragments or E Gel with SYBR for blotting see page
50. E Gel agarose gels are available from Invitrogen The recommended DNA marker for each gel type and ordering information is provided on pages 33 45 71 57 97 E Gel iBase USB Mini Cable Invitrogen Cat no G6300 is used to download firmware upgrades for the E Gel iBase Power System from the Invitrogen website The E Holder Platform is used to hold an E Gel 48 or 96 gel in place for robotic loading and is available from Invitrogen Invitrogen Cat no EH 03 The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to electrophorese E Gel agarose gels The gel knife Invitrogen Cat no EI9010 is used to open E Gel EX cassettes for excision of DNA fragments or blotting The E Gel Opener is a device specifically designed to open E Gel single comb and double comb cassettes excluding E Gel EX cassettes for excision of DNA fragments or blotting Ordering information is provided below Product Quantity Catalog no E Gel Opener 1 G5300 01 E Gel Opener Replacement Blades 10 G5350 10 The E Editor 2 02 software is available FREE of charge with the purchase of any E Gel 48 or 96 gels and related equipment The software may be downloaded at www invitrogen com egels Continued on next page 1X Accessory Products Continued Safe Imager Transilluminators Loading Buffers The E Gel Safe Imager Real time Transilluminator and Safe Imager Blue Li
51. E Gel 48 96 gels into any empty wells Total Volume Gel Type Second Step E Gel single comb gel 10 ul 10 pl E Gel double comb gel 10 ul 10 ul E Gel EX agarose gel 10 ul 10 pl E Gel 48 gel 5 ul 10 ul E Gel 96 gel 10 ul 10 pl E Gel SizeSelect agarose gel 10 ul 10 ul E Gel with SYBR Safe 10 ul 10 ul Loading buffer is required for the Two Step Loading method for E Gel agarose gels Mix the required amount of DNA with the loading buffer see below The total volume of the DNA sample and loading buffer should not exceed the volume listed for the second step see table above We recommend using a loading buffer with the following formulation in its final concentration E Gel agarose gels E Gel CloneWell EX and SizeSelect gels e 10 glycerol or 6 e 10 glycerol or 6 Ficoll 400 Ficoll 400 e 10mM Tris HCl pH 7 5 e 1mM EDTA e 0 005 bromophenol blue e 0 00576 xylene cyanol FF If using 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer from Invitrogen page viii dilute this buffer 50 to 200 fold and add 10 glycerol Continued on next page Two Step Loading of E Gel Agarose Gels Continued Two Step Loading Method High Salt Samples All wells in the gel must be loaded with either sample or water Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Load deionized water into each well include wells for sample molecular weight marker and empty
52. Gel SizeSelect Agarose Gels eene 83 Sample Preparation for E Gel SizeSelect Agarose Gels essere 83 Kun Time Estimation for Size5elect Agarose Gels aricii een ath reto tar nau ertet Rete 85 Loading E Gel SizeSelect Ap arose Gelsene eare oai eas ola Pct coses tod Donee oia 86 Running E Gel SizeSelect Agarose Gels eee eese eeesee eee tetn tente tinte etate tant nonut 89 Visualizing E Gel SizeSelect Agarose Gels eerte then tnnt tntn ttti tnt tnnt 92 Quantitation of DNA Isolated from E Gel SizeSelect Agarose Gels sss 93 TOU LE SIV OO CIV E 94 Electrophoresis of E Gel 48 96 Gels tetris inest strani 96 Sample Preparation for E Gel 48 96 Gels asus conto ioter P Se Pais Do deseas ies pea pia tu ee ecita deos 96 Loadims Bel 40 and 96 GelSEsmdbeei tias pic subdi Seam deii dieit ht dag uad idee DU 98 Rutitinp Biel 25 3nd 96 Gels atate a tion kanes CD rn MORS ved E dabo ieu di La rayeteaueses 103 E base Quick Reference Guide note ide iidem epa tiui dei pua UE 105 Usine BE Holdetr JPISUOEBEs aei spain it tipa b ideale a pali ci iu Pase M Lb adsunt biu ap bauen 107 Visualizing E Gel 96 with SYBR Safe Agarose Gels sesenta 108 Restilts itl E Geb 949 Gels d ostensae tox eid ea o Rope ea bo betont uncut di dete adid 109 Results with E Gel 96 GEE renas a veut esM Dados aM nasi ed Ou Pe ove 112 Using E Bditor 2 02 SO
53. Gel single comb and double comb agarose gels page 122 for details Power Supply comb wells underneath Top Bottom E Gel Opener Introduction The E Gel Opener is an easy to use device specifically designed to open any E Gel single comb double comb or E Gel with SYBR Safe cassette for staining excision of DNA fragments or for blotting Important Do not use the E Gel Opener to open the E Gel 48 or 96 cassettes The E Gel 48 or 96 cassette cannot be opened Description of The E Gel Opener consists of an anodized aluminum platform housing two Opener recessed steel blades one which is stationary and one which is movable The blades are brought into contact with the E Gel cassette by turning the large knob clockwise see Figure 1 1 Blade e E J P S Table edge La e Before using the E Gel Opener for the first time we recommend that you M Qu practice opening a few used E Gels to familiarize yourself with the process a E Practice on E Gels that will not be used further for preparative purposes e Electrophoresis must be complete before opening the E Gel We recommend that you place the E Gel on the transilluminator and photograph the gel before proceeding further If you plan to isolate DNA from the E Gel we recommend that you open the gel and excise the gel fragment immediately after electrophoresis as bands will diffuse within 20 minutes If you plan to blot the ge
54. Invitrogen E Gel Technical Guide General information and protocols for using E Gel pre cast agarose gels Version K December 12 2008 25 0645 ii Table of Contents A DOIG OF COMBCI NES M iii la CLIT VU E vi General PEORIA OM 5 aep evi Despues cae e alae d sues umts n tos uu brand temen sidus ite det aset vii ACCESSO PTOL UCI S eaa Eh renum echelon ous viii INEROGUCUION meer 1 E Gel Electrophoresis GY SSI opi tao lacus fais onte Ebo uetus cit opaca ta est Pia loalueme soe cal 1 eie luae du M a meen re E E Tor ree eRe eee ee eee ene eee 2 Low Throughput E Gel Electrophoresis System sssssssssssesssssssessesessessssesessesessnsssnsssesasseseeeees 4 Low Thtoughput E Ge Well FOrmmats disent pe ohio iaa 5 E Gel gt 1BAse POW Cr S ySIITLen qae Proident E Mand academia et eit ipd sta aci 6 PGE Power Base rapin e p E 8 EGEF ODODeb d sien E uen E N EE N E a 9 Gd m 11 Medium Throughput E Gel Electrophoresis System esses eterne 12 Peek 48 A ar Ose Gels uus arias retten pedes Aot A Pa Roten gustu des i tum dra ies 13 High Throughput E Gel Electrophoresis System eee eese ttti tntnt ntn tntnt ne 14 BGC cire GEIS me canoer 15 Ee Bise Power UDP rei n ate testeq ictus Na A AR 16 Nucleic Acid Gel
55. Marker 350 ng 12 Low DNA Mass Ladder 470 ng 2 Agarose GP O CON A FF FP WN me Continued on next page Results using E Gel with SYBR Safe Agarose Gels continued Examples Using DNA samples run on an E Gel 2 with SYBR Safe are shown below recorded Different Imaging using different imaging methods Samples were loaded in a total volume of 20 ul Methods and visualized using the indicated transilluminator and filter Photographs were taken using the MiniBis photo documentation system from DNR Standard 312 nm UV Standard 312 nm UV Blue light transilluminator transilluminator transilluminator Long pass ethidium SYBR Safe photographic SYBR Safe photographic bromide filter filter filter ow ico aoa eD 2 Agarose GP 2 Agarose GP 12345678 9MDN B 123456789MUH B M 000000 RR 2 Agarose GP 123456789 RB pm ga tf p pam Lo M A aM m E Gel with SYBR Safe E Gel with SYBR Safe invitrogen e Invitrogen E Gel with SYBR Safe invitrogen rm o Sample Low DNA Mass Ladder 470 ng E Gel Low Range DNA Marker 350 ng 240 bp PCR product 500 ng 317 bp PCR product 700 ng 1 kb PCR product 100 ng 100 bp DNA Ladder 900 ng 50 bp DNA Ladder 700 ng 100 bp DNA Ladder 900 ng pUC19 EcoR I cut 50 ng pUC19 uncut 50 ng E Gel Low Range DNA Marker 350 ng Low DNA Mass Ladder 470 ng
56. NA Ladder Continued on next page Results using E Gel EX Agarose Gels for RNA Samples E Gel EX 1 Agarose Gel E Gel EX 2 Agarose Gel An example of mouse brain RNA samples and the Invitrogen 0 1 2 Kb RNA Ladder run on an E Gel EX 1 agarose gel is shown below Samples were loaded in a total volume of 20 ul and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec Sample RNA Ladder native RNA Ladder native heat RNA Ladder native heat Total RNA native Total RNA native Total RNA 65 C 5 minutes Total RNA 65 C 5 minutes Total RNA formamide 65 C 5 minutes Total RNA formamide 65 C 5 minutes RNA Ladder native heat RNA Ladder native b E Gel EX Agarose Gel invitrogen y An example of mouse brain RNA samples and the Invitrogen 0 1 2 Kb RNA Ladder run on an E Gel EX 2 agarose gel is shown below Samples were loaded in a total volume of 20 ul and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec Sample RNA Ladder native RNA Ladder native heat RNA Ladder native heat Total RNA native Total RNA native Total RNA 6
57. Pre running Using 1 iBase Power System 5 Open the package and remove the gel Do not remove the comb until you start loading the samples page 46 Slide the cassette into the two electrode connections on the E Gel iBase Power System Press on the left side of the cassette to secure it into the iBase The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED light will illuminate a steady red to show that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure om e Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the program PRE RUN 2 minutes using the Up Down A V buttons to change the program Press the Go button to pre run the gel The LED light changes to green light to indicate that the cassette is in the pre run mode After two minutes pre run stops automatically as indicated by a red light and a beeping sound Continued on next page 47 Loading E Gel with SYBR Safe Gels continued Pre running Using PowerBase v 4 Method of Loading Samples Loading E Gel 48 1 2 Plug the PowerBase v 4 into an electrical outlet using the adaptor plug Open the package containing the gel and insert the gel with the comb in place into the apparatus right edge first Press firmly at the top and bottom to seat the gel in the ba
58. Samples can be loaded directly into the wells if no loading buffer is used If you are using loading buffer mix the required amount of DNA with the loading buffer We recommend using a loading buffer with the following formulation in its final concentration E Gel agarose gels E Gel CloneWell and SizeSelect gels e 10mM Tris HCl pH 7 5 e 10mM Tris HCl pH 7 5 e 1mM EDTA e 1mM EDTA e 0 005 bromophenol blue e 0 00576 xylene cyanol FF If using 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer from Invitrogen page viii dilute this buffer 50 to 200 fold to obtain optimal results with E Gel agarose gels Continued on next page General Guidelines Continued DNA Ladders E Gel 1 Kb Plus Ladder Tracklt Ladders High Salt Samples DNA ladders can be used to estimate the size of fragments and to track the progress of a run Suggested ladders are listed in the description for running each type of gel The E Gel 1Kb Plus DNA Ladder is recommended for use with E Gel EX and E Gel SizeSelect gels as well as other E Gel precast gels If using TrackIt DNA ladders for molecular weight estimation do not use more than 2 ul in a total load volume of 20 ul TrackIt DNA ladders are not recommended for use with E Gel EX or E Gel SizeSelect agarose gels Important Samples containing 250 mM NaCl 100 mM KCI 10 mM acetate ions or 10 mM EDTA i e certain restriction enzyme and PCR buffers will cause lo
59. Select pre cast agarose gels provide a novel way to purify DNA bands and offer the following advantages e Saves time by not requiring additional gel purification steps after electrophoresis e Simplifies DNA recovery since purified DNA is removed directly from the well with a pipette e Improves cloning results by minimizing UV related DNA damage leading to more colony forming units than other cloning methods e Supplied as precast 0 8 E Gel CloneWell or 2 E Gel SizeSelect agarose gels in the familiar E Gel format allowing fast safe consistent and high resolution separation of small and large DNA fragments For details on E Gel CloneWell agarose gels see page 70 For details on E Gel SizeSelect agarose gels see page 83 E Gel iBase Power System E Gel iBase Power System The E Gel iBase Power System figure below is an easy to use automated device specifically designed to simplify electrophoresis of single comb or double comb E Gel agarose gels from Invitrogen The E Gel iBase is a base and a power supply all in one device The E Gel iBase Power System has an LCD display which shows information about the program selected and running time The display is located near the upper edge of the iBase Just below the display the E Gel iBase Power System has four buttons see image below e A Go button to start programs e A Mode button to toggle between programs minutes and
60. Stains Tor E Gel Agarose Gels usce aa E dateien R tenti 18 SY DI Sate DNA Goel eaa O E deli eed ded 19 Proprietary Fluorescent N cleic Acid Gel Stalb sos ote a a 21 Safe Imager Blue Light Transilluminators eesesseersreeresssrrsrereererserrrrereseerersrrrererierersarnrreresrererenrnerereees 22 Product SPeCIIICA ONS srana a E N 27 MICU OOS cripian aata iaa haa aaia paai aa aaa 30 General Guidelines iconic iori eo aaO ROGO AR RE OUO ED KDE ED RR aE 30 Electrophoresis of E Gel Agarose Gels raise T S R Oe 32 Sample Preparation for E Gel Agarose Gels oen eet t paie seen que E uua RUE 32 LoadmgsSmglecComb and Double Comb Gels onte stet peo et tn tta edens a p ondes seio dae 35 Running Single Comb and Double Comb G8lS isotope tuoi eR eph oniiir lbs tea ton ii 37 Results with E Gel 5imele Comb Gels pao Opin a Qc ted A NE 40 R sults with E Gel Double Comb Gel5 pasada na n A A 42 Troubles ho ln isni a a a a a 43 Electrophoresis of E Gel with SYBR Safe Gels c cscsssscessssessesesceseccesescessecessecersecersnsessnsessnsenees 44 Sample Preparation for E Gel with SY BR Safe eene pete Peter ehe ipso n aeep etnies etapa ote 44 Loadine Bel with XB Sale Gelgit Most inp ab vestes E 46 Roaming E Gel With SY BR Sale Gels auae a eda top R ust up eae c treten 49 Visualizing E Gelo with oy DR sale Agarose Gels echtes tti ns UR OU A tae NI In ire tdie ine 51 Results using E Gel with SYBR Safe Agarose Gels
61. a total volume of 20 ul and visualized on a standard 312 nm UV transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec o Sample High DNA Mass Ladder 130 ng E Gel High Range DNA Marker 200 ng 1 kb PCR product 100 ng 3 kb PCR product 200 ng 9 kb PCR product 200 ng 1 Kb plus DNA Ladder 500 ng 1 Kb plus DNA Ladder 500 ng pBR322 EcoR I cut 100 ng pBR322 uncut 100 ng E Gel with SYBR Safe 10 pUC19 EcoR I cut 50 ng invitrogen 11 E Gel High Range DNA Marker 200 ng 12 High DNA Mass Ladder 130 ng 1 2 Agarose GP 12345 6 8 90r it TESI O CON A FT MARAON E An example of DNA samples run on an E Gel 2 with SYBR Safe is shown below Samples were loaded in a total volume of 20 ul and visualized on a standard 312 nm UV transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec p D Sample Low DNA Mass Ladder 470 ng E Gel Low Range DNA Marker 350 ng 240 bp PCR product 500 ng 317 bp PCR product 700 ng 1 kb PCR product 100 ng 100 bp DNA Ladder 900 ng 50 bp DNA Ladder 700 ng 100 bp DNA Ladder 900 ng pUC19 EcoR I cut 50 ng E Gel with SYBR Safe 10 pUC19 uncut 50 ng Invitrogen 11 E Gel Low Range DNA
62. ackIt 1 Kb Plus DNA Ladder 10488 085 34 Loading Single Comb and Double Comb Gels Introduction Pre running E Gel Pre running Using iBase Power System After you have prepared your samples you are ready to proceed with electrophoresis Instructions are provided below to load and run E Gel single comb and double comb gels using the E Gel iBase Power System or E Gel PowerBase v 4 for electrophoresis For details on using the E Gel agarose gels with the E Gel Base see page 122 You must first pre run the E Gel agarose gels for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your bands Each E Gel cassette is supplied individually wrapped and ready for use To set up and use an E Gel agarose gel using an E Gel iBase Power System follow the instructions below If you are using a PowerBase v 4 see next page 1 Attach the power cord of the E Gel iBase to the power inlet and then to the electrical outlet Use only properly grounded AC outlets and cords Open the package and remove the gel Do not remove the comb until you start loading the samples page 36 Slide the cassette into the two electrode connections on the E Gel iBase Power System Press on the left side of the cassette to secure it into the E Gel iBase The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED ligh
63. an ethidium bromide and Gain Setting X stained gels As a result a shorter exposure time or lower gain setting may be necessary 92 Quantitation of DNA Isolated from E Gel SizeSelect Agarose Gels Quantitation Size and quantity of recovered DNA can be assessed by gel electrophoresis For accurate quantitation of DNA recovered from SizeSelect gels for applications such as next generation sequencing libraries we recommend performing qPCR For fluorometric quantitation we recommend using the Qubit fluorometer Invitrogen Cat no Q32857 with the Quant iT dsDNA HS Assay Kit Invitrogen Cat nos 32851 or Q32854 from Invitrogen An accurate working range can accommodate up to 40 ng of DNA in the final Qubit reaction mixture For spectrophotometric quantitation of recovered DNA we recommend buffer exchange using the Invitrogen PureLink PCR Micro kit Invitrogen Cat nos K310010 or K310050 93 Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel SizeSelect agarose gels No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before cassette the specified expiration date E Gel SizeSelect cassette is Remove cassette and reinsert a steady red light not inserted properly intoa illumi
64. ands Do not use this device in damp areas or while standing on damp floors The Safe Imager Blue Light Transilluminator is supplied with an international power cord This power cord has a universal transformer compatible with either 110 V or 220 V electrical outlets and a selection of plug adaptors so that it may be used with any electricity supply Use only the power cord supplied with the Safe Imager transilluminator to power the device Attach the supplied power cord to the Safe Imager transilluminator at the back of the device Plug the other end of the power cord into a properly grounded electrical outlet ensuring the correct plug adaptor is attached Always disconnect the Safe Imager transilluminator from the electrical outlet before cleaning the device The Safe Imager blue light transilluminator does not produce UV light However the intense blue light emitted by the Safe Imager may promote macular degeneration upon prolonged exposure especially in those prone to such problems e g people with fair complexion and blue eyes nutritional or endocrine defects or those who are aging Use the Safe Imager amber filter unit or Safe Imager viewing glasses provided with this device to protect your eyes The amber filter unit and viewing glasses are for viewing stained gels using the Safe Imager blue light transilluminator The amber filter unit is NOT a safety screen for UV emission and will NOT protect your eyes when viewing ge
65. aptor included with the PowerBase Input and Output supplied by the adaptor are shown in the table below US and Canada 110 120 V AC 60 Hz 12 V DC 880 mA Europe 220 240 V AC 50 Hz 12 V DC 880 mA The specifications for E Gel Base are listed below Dimensions 12 5x 13x 3 5 cm Weight 3 18 oz 90 g Temperature Ambient 15 C to 40 C 29 Methods General Guidelines Introduction Materials Needed General Guidelines Loading E Gel Agarose Gels Loading Buffer 30 For optimal results follow these general guidelines for preparing your DNA sample For specific details related to running each type of E Gel agarose gel refer to the section for that particular gel type e DNA sample e Loading buffer optional e Molecular weight markers e Run gels stored at room temperature e Keep samples uniform and load deionized water into empty wells e Load gel within 15 minutes of opening the pouch e Run gel within 1 minute of loading samples DNA samples are loaded in E Gel agarose gels using a One Step Loading or Two Step Loading method The One Step Loading method is the standard method for loading E Gel agarose gels The Two Step Loading method is an optional method that is only necessary if the One Step Loading method produces fuzzy or indistinct bands or the gel has been removed from its plastic pouch for an extended period of time see Appendix page 126 for details Loading buffer is optional
66. aring your DNA sample as described in this section Materials Needed DNA sample e Loading buffer with diluted tracking dye optional e Molecular weight markers page 57 Amount of DNA For optimal results refer to the following tables when determining the amount of sample to run on an E Gel EX agarose gel If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution E Gel EX 1 agarose gel Single DNA Multiple DNA Optimal Sample Maximum Band Bands Amount Sample Amount 1 100 ng 1 50 ng band E Gel EX 2 agarose gel Single DNA Multiple DNA Optimal Sample Maximum Band Bands Amount Sample Amount 1 300 ng 1 100 ng band 500 ng Total Sample The recommended total sample volume for E Gel EX is 20 pl Volume Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells The proprietary fluorescent nucleic acid stain in E Gel EX agarose gels is more Note sensitive than ethidium bromide For cloning be sure to load enough DNA for your application as quantities of DNA that are sub optimal for cloning can still produce a strong signal Preparing Prepare your samples by adding deionized water or TE to the required amount Samples of DNA to bring the total sample volume to 20 ul For
67. at the 2 minute pre run has started 5 Atthe end of the pre run current will automatically shut off The flashing green light will change to a flashing red light and the PowerBase will beep rapidly 6 Press and release either button to stop the beeping you will hear only one beep The light will change from a flashing red light to a steady red light The E Gel agarose gels are designed for loading samples manually or using a multichannel pipettor We recommend the following methods of sample loading based on the gel type Gel Type Method of Loading E Gel single comb Manual E Gel double comb Manual or multichannel pipettor All wells in the gel must contain sample or water Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Remove the comb from the E Gel gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette 2 Load samples in 20 ul volume into the wells Load 20 ul of water into any remaining empty wells 3 Load 100 250 ng of the appropriate molecular weight markers page 33 Running Single Comb and Double Comb Gels Introduction Electrophoresis Using iBase Power System Speed Runs Using iBase After you have loaded your samples you are ready to proceed with electrophoresis Instructions are provided below to run E Gel agarose g
68. at which it was stopped reinsert the gel and press and release the Go button The light changes to steady green and the LCD display shows the count down time It is also possible to change the remaining run time but not the program as described on the previous page before continuing the run To cancel the rest of the interrupted run press and hold the Go button for a few seconds The LCD display will reset and the base will return to Ready Mode If desired you can then select a new program or run time as described on the previous page and rerun the gel E Gel EX agarose gels can only be used once Do not re use them E Gel EX agarose gels already contain a proprietary DNA gel stain and therefore do not have to be stained after the electrophoresis run Continued on next page Visualizing E Gel EX Agarose Gels Introduction Viewing E Gel EX Agarose Gels Imaging E Gel EX Agarose Gels Required Filters Important Exposure Time and Gain Setting Bound to nucleic acids the proprietary DNA gel stain in E Gel EX agarose gels has fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate see below View E Gel EX agarose using these instruments e Blue light transilluminator The E Gel Safe Imager Real time Trans illuminator
69. ble for use with E Gel with SYBR Safe e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel with SYBR Safe using a CCD camera or a laser based scanner For photographing gels refer to page 125 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel with SYBR Safe While yielding similar sensitivities to ethidium bromide SYBR Safe is somewhat dimmer yet with a lower background than ethidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary 5 Results using E Gel with SYBR Safe Agarose Gels Introduction E Gel 1 2 with SYBR Safe E Gel 2 with SYBR Safe 52 On this page we display typical results using E Gel with SYBR Safe agarose gels 1 2 and 2 On the next page examples of the same E Gel 2 with SYBR Safe recorded with different imaging methods are shown An example of DNA samples run on an E Gel 1 2 with SYBR Safe is shown below Samples were loaded in
70. continuously automatically stops at 00 121 Using E Gel Base Introduction Pre run with an E Gel Base Running E Gel on an E Gel Base 122 Instructions are provided below to perform electrophoresis of E Gel with SYBR Safe E Gel single comb gels and double comb gels with the E Gel Base previously available from Invitrogen For instructions on using the E Gel PowerBase see page 35 Note You will need a power supply for electrophoresis with an E Gel Base You must first pre run the E Gel agarose gel for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your DNA fragments Each E Gel cassette is supplied individually wrapped and ready for use To set up and use an E Gel gel follow the instructions below 1 Open the package containing the gel and insert the gel with the comb in place into the apparatus right edge first 2 Press firmly at the top and bottom to seat the gel in the base You should hear a snap when it is in place The Invitrogen logo should be located at the bottom of the base close to the positive pole See the diagram below Power Supply 7 7 a pole 8 8 e Black CAP comb wells underneath Top Bottom 3 Connect electrical leads from the base unit to the power supply Pre run the gel with the comb in place for 1 2 minutes at 60 70 V or 40 50 mA Do not exceed 2 minutes Turn off the power suppl
71. d at room temperature Store the E Gel Base E Gel iBase E Gel PowerBase and E Base at room temperature Avoid storing or using any electrophoresis bases at 4 C e Some E Gel agarose gels contain ethidium bromide a known mutagen The concentration of ethidium bromide in each gel ranges from 0 1 to 0 3 ug ml All E Gel agarose gels contain 0 055 Proclin added as a preservative apart from E Gel 48 4 gels which contain 0 01 Thimerosal Each gel is provided in a sealed package so you are protected from exposure As a precaution always wear gloves and protective clothing when handling the gel e Dispose of used E Gel agarose gels containing ethidium bromide E Gel EX and E Gel SizeSelect agarose gels as hazardous waste e Avoid overexposure of skin and eyes when using UV light e Avoid overexposure of eyes when using intense blue light e Avoid touching the gel during electrophoresis Vil Accessory Products vili E Gel Agarose The following E Gel agarose gels are available from Invitrogen Ordering Gels information is described below Product Quantity Catalog no E Gel CloneWell Gels E Gel CloneWell 0 8 SYBR Safe Gels 18 Pak 18 gels G6618 08 E Gel CloneWell 0 8 SYBR Safe Gel and iBase Starter Kit 18 gels E Gel iBase Power System Safe G6500ST Imager Blue Light Transilluminator and G6500STEU E Gel High Range DNA Marker G6500STUK E Gel Single Comb Gels E Gel 1 2 with SYBR Saf
72. d light will change to a green light and the digital display will show the count down time while the run is in progress to electrical outlet electrode connection E Gel 96 gel A electrode Sahai connection power button timer button Mother E Base digital timer 9 t LED display If you are using a Daughter E Base press and release the pwr prg button located on the lower right corner of the Daughter E Base Mother E Base To add to the run time while the run is in progress press the time button to select the desired time and then release the time button To interrupt or stop a run in progress see next page The Mother E Base or Daughter E Base will signal the end of the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute At the end of the run the digital display will show the original time setting not any time change that was made during the electrophoresis The digital display will also show the elapsed time up to 19 minutes with a negative sign since the end of the run Press and release the pwr prg button to stop the beeping The light will turn to a steady red and the digital display will show the last time setting Remove the gel cassette from the Mother E Base or Daughter E Base You are now ready to capture an image of the gel Note The bands in the gel will diffuse within 20 40 minutes Continued on next page
73. e Starter Kit 6 gels and E Gel PowerBase G6206 01 E Gel 1 2 with SYBR Safe 18 gels G5218 01 E Gel 2 with SYBR Safe Starter Kit 6 gels and E Gel PowerBase G6206 02 E Gel 2 with SYBR Safe 18 gels G5218 02 E Gel EX 1 Starter Kit 10 gels E Gel iBase Power System Safe G6511ST Imager Blue Light Transilluminator and G6511STUK E Gel 1 Kb Plus DNA Ladder G6511STEU E Gel EX 1 10 Pak 10 gels G4010 01 E Gel EX 1 20 Pak 20 gels G4020 01 E Gel EX 2 Starter Kit 10 gels E Gel iBase Power System Safe G6512ST Imager Blue Light Transilluminator and G6512STUK E Gel 1 Kb Plus DNA Ladder G6512STEU E Gel EX 2 10 Pak 10 gels G4010 02 E Gel EX 2 20 Pak 20 gels G4020 02 E Gel SizeSelect 2 10 Pak 10 gels G661002 E Gel SizeSelect 2 Starter Kit 10 gels E Gel iBase Power System Safe G6612ST Imager Blue Light Transilluminator and G6612STEU 50 bp DNA Ladder G6612STUK E Gel 0 8 with Ethidium Bromide Starter Pak 6 gels and E Gel PowerBase G6000 08 E Gel 0 8 with Ethidium Bromide 18 Pak 18 gels G5018 08 E Gel 1 2 with Ethidium Bromide Starter Pak 6 gels and E Gel PowerBase G6000 01 E Gel 1 2 with Ethidium Bromide 18 Pak 18 gels G5018 01 E Gel 2 with Ethidium Bromide Starter Pak 6 gels and E Gel PowerBase G6000 02 E Gel 2 with Ethidium Bromide 18 Pak 18 gels G5018 02 E Gel 4 with Ethidium Bromide 18 Pak 18 gels G5018 04 E Gel Double Comb Gels E Gel 0 8 6 double comb with Ethidi
74. e the E Gel cassette begins to lift off the surface of the platform do not continue to tighten the knob as you will damage the E Gel Unscrew the knob and remove the E Gel The E Gel cassette fits snugly in the recessed groove and you may have to carefully work the cassette from the housing Turn the E Gel 90 and re insert the gel cassette into the Opener so that the two remaining sealed sides can be opened Repeat Step 2 to open the remaining two sides of the E Gel Stop turning the knob when you see the top of the E Gel cassette begins to lift off the gel Unscrew the knob and carefully remove the E Gel cassette The 4 sides of the cassette should be unsealed If not repeat Steps 2 5 as necessary Remove the E Gel and set the opened cassette on your bench If you plan to blot the gel do not pick up the gel from the cassette Lift off the top of the gel cassette Place the blotting membrane on the gel and pick up the cassette with the gel and membrane Flip the gel and membrane out of the cassette onto your gloved hand and then flip the gel and the membrane directly onto your wet blotting paper If you plan to purity DNA from the gel lift off the top of the gel cassette and excise the gel fragment Transfer the gel slice to a microcentrifuge tube Discard E Gel agarose gels with ethidium bromide as hazardous waste SYBR Safe stain is not classified as hazardous waste under US Federal regulations but contact your safety
75. e the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program 7 Remove the E Gel cassette from the E Gel iBase You are now ready to proceed to imaging or any other application with the gel Continued on next page Running Single Comb and Double Comb Gels continued Electrophoresis Using PowerBase v 4 Note Opening the E Gel Cassette 1 On the E Gel PowerBase v 4 choose between a 30 minute run for single comb gels and a 15 minute run for double comb gels For the 30 minute run press and release the 30 min button to start the 30 minute electrophoresis run The light will change to a steady green light For the 15 minute run press and release the 15 min button A steady blue light appears to indicate the beginning of the 15 minute run Note The actual running time of the E Gel gel may vary between 15 17 minutes for double comb gels and 30 33 minutes for single comb gels 2 Current through the E Gel gel automatically shuts off at the end of each run The E Gel PowerBase v 4 signals the end of the run with a flashing red light and rapid beeping 3 Press and release either button to stop the beeping The light will turn to a steady red light 4 Atthe end of the run remove the gel cassette from the power unit and analyze your results using a UV transilluminator E Gel agarose gels can only be used once Do not re use them
76. e with E Gel SizeSelect agarose gels Refer to the next section for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager Blue Light Transilluminator See page 22 for details Blue light transilluminators available from other manufacturers are also compatible for use with E Gel SizeSelect agarose gels e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm e View SizeSelect agarose gels using amber filter or amber viewing goggles e For imaging with a laser based scanner verify the system has an excitation source compatible with the proprietary dye Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources in combination can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Imaging E Gel Photograph E Gel SizeSelect agarose gels using a CCD camera or a laser SizeSelect based scanner Agarose Gels Required Filters For photographing gels refer to page 125 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for portant photographing E Gel SizeSelect agarose gels Exposure Time E Gel SizeSelect agarose gels have greater sensitivity th
77. eWell Agarose Gels Required Filters Important Exposure Time and Gain Setting Bound to nucleic acids SYBR Safe DNA gel stain has fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm To minimize DNA damage use a blue light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate see below View E Gel CloneWell agarose gels using a Blue light transilluminator The E Gel Safe Imager Real time Trans illuminator and Safe Imager Blue Light Transilluminator see page x are designed specifically for use with SYBR Safe stained DNA gels Refer to page 22 for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager Blue Light Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel with SYBR Safe Note UV light sources can lead to reduced cloning efficiencies Photograph E Gel with SYBR Safe using a CCD camera or a laser based scanner For photographing gels refer to page 125 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel with SYBR Safe While yielding similar sensitivities to ethidium bromide SYBR Safe is somewhat dimmer yet with a lower background than e
78. eal time Transilluminator should be used with the power cord supplied your starter kit or with the E Gel iBase Power System This power cord has a universal transformer compatible with 90 V to 220 V Only these power cords should be used to power the device Attach the power cord to the E Gel Safe Imager Real time Transilluminator at the back of the device Plug the other end of the power cord into a properly grounded electrical outlet ensuring the correct plug adaptor is attached Always disconnect the E Gel Safe Imager Real time Transilluminator from electrical outlet before cleaning device The E Gel Safe Imager Real time Transilluminator does not produce UV light however it does utilize an intense blue light for viewing gels It should be noted that published literature has identified blue light as a possible risk factor for macular degeneration however no clinical studies have been published Therefore the E Gel Safe Imager amber filter unit or E Gel Safe Imager viewing glasses provided with this device should always be used to protect your eyes while viewing gels Note The amber filter unit is NOT a safety screen for UV emission and will NOT protect your eyes when viewing gels on UV transilluminators And although the viewing glasses do block UV light they are not designed for use as UV safety glasses Do not leave the E Gel Safe Imager Real time Transilluminator switched on for extended periods of time After v
79. econfigures the wells of an E Gel 48 and E Gel 96 gel into a side by side format for easy comparison and analysis You can reconfigure gels that were scanned in the original gel cassette or gels that were removed from the cassette You can also group the images of multiple gels loaded from a 384 well microtiter plate into a single image with a layout corresponding to that of the original plate Capture an image of the gel as described below and then use the E Editor 2 02 software to e Align and arrange the lanes in the image e Save the reconfigured image for further analysis e Copy and paste selected lanes or the entire reconfigured image into other applications for printing saving e mailing and or publishing on the Web Use an appropriate gel documentation system to capture a digital image of the gel When imaging the gel should be properly aligned i e not at an angle and gel features should be clear and distinct Proceed to Downloading Software E Editor 2 02 software can be downloaded for free from the Invitrogen website Go to www invitrogen com egels and follow the instructions to download the software and user manual 115 Troubleshooting Troubleshooting The table below provides some solutions to possible problems you might encounter during the electrophoresis of E Gel 48 and 96 agarose gels To troubleshoot problems with single and double comb E Gel see page 43 No current Daughter E Base used Do not u
80. ections on the E Gel iBase Power System Press firmly at the top and bottom of the left side of the cassette to secure it into the iBase The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED light will illuminate a steady red to show that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure Important Do not pre run E Gel EX agarose gels The E Gel EX agarose gels are designed for manual loading of samples All wells in the gel must be loaded with either sample or water Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 2 Load 20 ul of sample per sample well page 56 for sample preparation Load 20 ul 100 250 ng of the appropriate molecular weight markers page 57 Load 20 ul of water into any remaining empty wells Continued on next page Running E Gel EX Agarose Gels Introduction After you have loaded your samples you are ready to proceed with electrophoresis Instructions are provided below to run E Gel EX agarose gels using the E Gel iBase Power System Electrophoresis 1 Using iBase Power System Toggle between program minutes and seconds on the iBase by pressing the Mode button until the program blinks Select the program E Gel EX program 7 using the Up Down A V buttons to change the program Default run time for E Gel EX is 10
81. ectrophoresis of two or more E Gel 48 E Gel 96 E PAGE 48 or E PAGE 96 gels available from Invitrogen Note The Daughter E Base does not have an electrical plug and cannot be used without a Mother E Base See next page for a diagram of the bases Each Mother E Base has a pwr prg power program button right side and a time button left side on the lower right side of the base The lower left side of each Mother E Base contains a light LED and a digital display The gel cassette is inserted into the two electrode connections The Mother E Base is connected to an electrical outlet with the electrical plug The E Base is pre programmed with 2 programs specific for each gel type as described below Program Gel Type Run Parameters EG E Gel 96 Time 12 minutes EP E PAGE 96 Time 14 minutes EG E Gel 48 1 and 2 Time 20 minutes EG E Gel 48 4 Time 17 minutes EP E PAGE 48 Time 23 minutes Mother E Base to electrical outlet t electrode E _4 electrode gt pwr prg Sc button time button Continued on next page E Base Power Supply Continued Daughter E Base gt E Holder Platform E Editor 2 02 Software The Daughter E Base is similar to the Mother E Base except the Daughter E Base does not have an electrical cord and cannot be connected to an electrical outlet The Daughter E Base is connected to a Mother E Base or to another Daughter E Base already
82. efault time setting 12 minutes for EG 14 minutes for EP or last time setting Count down time Negative time display 00 to 19 minutes Negative time display 00 to 19 minutes Flashing time display Continued on next page 105 E Base Quick Reference Guide Continued Mode Action Sound Light Digital Display Return to Press and Steady red Last time Ready mode release the setting after an pwr prg button automatic stop Restart after Press and Steady Count down a manual release the green time stop pwr prg button Return to Ready mode after a manual stop Failure Press and hold the pwr prg button Press and hold With gel cassette in steady red Without gel cassette no light With gel cassette in last time setting Without gel cassette last program setting Flashing ER pwr prg button loud for 2 seconds beeping No cassette Time setting Program setting and remove gel from the base With gel cassette in Press and release the time With and without gel cassette Press and hold the time button Press and release the pwr prg button when no cassette 1s inserted into the E Base to select the desired program With gel cassette steady red With gel cassette in steady red Without gel cassette no light No light EP last program used EP or EG Time increases by 1 minute increments lime increases co
83. el SizeSelect pre cast agarose gels offer the following advantages e Detection sensitivity to 1 ng band of DNA e Compatibility with blue light transillumination to reduce DNA damage that lowers cloning efficiency SYBR Safe DNA Gel Stain Introduction Safety Features of SYBR Safe Disposal of SYBR Safe SYBR Safe DNA gel stain has been specifically developed for reduced mutagenicity making it safer than ethidium bromide for staining DNA in agarose gels The detection sensitivity of E Gel with SYBR Safe stain is similar to that of E Gel containing ethidium bromide DNA bands stained with SYBR Safe DNA gel stain can be detected using a standard UV transilluminator a visible light transilluminator or a laser based scanner e SYBR Safe DNA gel stain is not classified as hazardous waste under US Federal regulations e SYBR Safe stain meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations though E Gel with SYBR Safe generally does not generate liquid waste e SYBR Safe DNA gel stain does not induce transformations in primary cultures of Syrian hamster embryo SHE cells In contrast ethidium bromide tests positive in the SHE cell assay consistent with its known activity as a strong mutagen e SYBR Safe stain does not cause mutations in mouse lymphoma cells at the TK locus nor does it induce chromosomal aberrations in cultured human peripheral blood ly
84. ell 19 42 43 PCR product 317 bp 100 ng well 111 Results with E Gel 96 Gels 2 E Gel 96 with Results obtained using a 2 E Gel 96 with SYBR Safe gel are shown in the SYBR Safe figure below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged on a Safe Imager Blue Light Transilluminator You can vary the amount of markers loaded to improve gel imaging Note The wells of the E Gel 96 gel are staggered DNA bands migrate between adjacent wells in the row below For example the bands of lane A2 will migrate between wells B1 and B2 The box highlights a lane The gel contains the following samples Lane Sample 12 L1 25 ng 10 kb fragment Fermentas Cat no SM1751 3 4 9 10 25 ng 300 ng fragment Fermentas Cat no 5M1621 5 6 7 8 25 ng of pUC18 Fermentas Cat no SD0051 2 68 kb cut with EcoR I Invitrogen Cat no 15202 013 M 8 ul of Low Range Quantitative marker Invitrogen Cat no 12373 031 112 Results with E Gel 96 Gels Continued 1 E Gel 96 Gel Results obtained using a 1 E Gel 96 gel are shown in the figure below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS120 system You can use a mini transilluminator to view the bands You can vary the amount of markers loaded to improve gel imaging Note The wells of the E Gel 96 gel are staggered DNA bands
85. els using the E Gel iBase Power System or E Gel PowerBase v 4 For details on using the E Gel agarose gels with the E Gel Base see page 122 1 Toggle between program minutes and seconds on the E Gel iBase by pressing the Mode button until the program blinks Use the Up Down A V buttons to select the proper program Gel Type Program Default Run Maximal Time Run Time E Gel 0 8 1 296 2 RUN E Gel 26 minutes 40 minutes E Gel 4 RUN E Gel 4 30 minutes 40 minutes E Gel double comb RUN E Gel DC 13 minutes 20 minutes 0 8 2 Note For 0 8 1 2 and 2 E Gels the E Gel iBase Power System has the option of speed runs if you want quick results See below for more information 2 If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons up to the maximal run time indicated in the table 3 Take out the comb and load your samples Be sure to load molecular weight markers and add water to any empty wells 4 To start electrophoresis press the Go button a green light illuminates to show that the run is in progress The LCD displays the count down time while the run is in progress 5 The run stops automatically when the programmed time has elapsed The E Gel iBase signals the end of the run with a flashing red light and rapid beeping for 30 seconds followed by a single beep every minute The LCD displays Run Complete Pre
86. empty wells Gel was not For best results run the gel within 15 minutes of sample electrophoresed loading immediately after If you cannot run the gel immediately after sample sample loading loading use the Two Step Loading method page 126 Expired gel used Use properly stored gels before the expiration date Longer electrophoresis Longer run times cause an increase in the current run time or high resulting in poor band migration or melted gel Do not run current during the run the gel longer than recommended time for each gel type Sample leaking Sample is overloaded Load the recommended sample volume per well from the wells Use Use the Two Step Loading method page126 Two Step Use the Two Step Loading method page126 method page 126 Wells damaged during Remove the comb gently without damaging the wells comb removal Failure Mode Defective cassette Disconnect the base and replace gel cassette with a fresh indicated by gel cassette Press and release the power button or Go continuous rapid button to return to Ready Mode beeping and Cold cassette or Use a cassette stored at room temperature Avoid storing improper operating gel cassettes at 4 C Use E Gel iBase PowerBase and E conditions Gel Base at room temperature 20 25 C Cassette Missing Hold Go to Reset or a steady red light 43 Electrophoresis of E Gel with SYBR Safe Gels Sample Preparation for E Gel with SYBR Safe Introd
87. ercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 57
88. f the Mother E Base and Daughter E Base should be kept free of contaminants To clean disconnect bases from power source and wipe clean with a dry cloth Do not attempt to open the Mother E Base or Daughter E Base To honor the warranty bases should only be opened and serviced by Invitrogen Introduction E Base Quick Reference Guide A quick reference guide for operating the Mother E Base and Daughter E Base is provided below Operating modes and electrophoresis runs are described Mode Action Sound Light Digital Display Base plugged in Ready with no current flowing through gel End of run Run ends after an external power failure Pause manually end the run Mother E Base connected to an electrical outlet Gel cassette inserted into a base Press and release the pwr prg button Automatic Automatic Press and release the pwr prg button during the run Continuous beeping for 2 minutes followed by a single beep every minute Continuous beeping for 2 minutes followed by a single beep every minute No light if a cassette is not inserted or red light if a cassette is inserted Steady red Steady green Flashing red until the time button is pressed Alternating red and green With gel cassette in steady red Without gel cassette no light Without gel cassette EP last program used EP or EG With gel cassette in last time setting D
89. ftWafe guasto teg oes de et n best ope sad eru oi ta ou eil R e dra tunt Rude 115 TEOUDISSIOOLIng serrian ctt isque bd dicto bte ete Dubtu d bees su ustubisa ts 116 ADBSDODCUS ust EMI MM Mi IM MEM MEME 118 Using E Gel 96 Mother Daughter B386 ss ai eet cie ead Ut vacas tate aaa 118 E Gel 96 Mother Daughter Base Quick Reference Guide sese 120 Une T Gel D3SOstocnos qeu ann E ute dM eat eer ome errere etre reer ere 122 Downloading Firmware Upgrades for the E Gel iBase sss 123 Parameters Tor E Gel iBase DtOPEatisstoupesse tatis Mas T istuc Pee eva e 124 Filtet Selecton GUIOG odo ect tu dut Base lh clr m a edd tem reins ten 125 Two Step Loading of E Gel Agarose Gels eese ene nennen nes 126 Explanation of Symbols and Warning Sitat t me hl terti pat i a boi Een banda eet bM E ance Estes 128 Technical SUD POT ts uo ood tistetebetu tette A iot ute don lu eei Loue stead 131 Parc haser Notilica tiossaan E baie piis Pa teque nit tube aret E EU cead ite e vede idis 132 Warranty E Gel Equipment Warranty vi Invitrogen warrants that E Gel iBase E Gel Powerbase v 4 Mother E Base Daughter E Base and E Holder will be free from defects in material and workmanship for a period of one 1 year from date of purchase If a defect is present Invitrogen will at its option repair replace or refund the purchase price of this product at no charge to you provided it is retu
90. gels provide a novel way to purify DNA bands with no purification necessary for downstream applications such as cloning E Gel CloneWell gels contain the safer and environmentally friendly SYBR Safe DNA gel stain enabling visualization of bands with a blue light transilluminator thus minimizing DNA damage For optimal results follow the guidelines for preparing your DNA sample as described in this section e DNA sample e Loading buffer optional e Molecular weight markers page 71 For best results use 50 200 ng DNA per band though 20 400 ng per band is acceptable Load up to 500 700 ng of samples containing multiple bands per well If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution Load 20 25 pl total sample volume per well Lower volumes may result in poor band shape Excess sample may cause well to well contamination Load 5 10 ul DNA molecular weight marker into the small middle well Note For best results keep all sample volumes uniform If you do not have enough samples to load all wells of the gel load an equal volume of deionized water into any empty wells Prepare your samples as described below 1 Prepare the samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 25 ul 2 Prepare the DNA molecular weight marker by adding deionized water to the req
91. ght Transilluminator are specifically designed for use with E Gel EX E Gel SizeSelect and SYBR Safe stained DNA gels See pages 20 21 for viewing options Product Quantity E Gel Safe Imager Real time Transilluminator 1 Device and Amber Filter E Gel iBase and E Gel Safe Imager Combo Kit US EU UK versions Pn E Gel Safe Imager Real time Transilluminator 1 kit Starter Kit for Cloning US EU UK versions Safe Imager Blue Light Transilluminator 1 Safe Imager Viewing Glasses 1 Safe Imager International Power Cord replacement 1 Safe Imager Amber Filter Unit replacement 1 Catalog no G6500 G6465 G6465EU G6465UK G65008T G6500STEU G6500STUK 537102 537 103 537104 537105 Loading buffers are optional for E Gel agarose gels The following loading buffers available from Invitrogen are suitable for use with E Gel agarose gels but should be diluted 50 200 fold before use Product Quantity 10X BlueJuice Gel Loading Buffer 10x 3x1ml TrackIt Cyan Orange Loading Buffer 3 x 0 5 ml TrackIt Cyan Yellow Loading Buffer 3 x 0 5 ml Catalog no 10816 015 10482 028 10482 035 Introduction E Gel Electrophoresis System Introduction Advantages of E Gel Agarose Gels Throughput Capacity The E Gel agarose gel electrophoresis system is a complete bufferless system for agarose gel electrophoresis of DNA samples The major components of the system are e E Gel pre cast agarose gels e
92. hoon 9200 9210 8600 8610 GE Healthcare 488 nm 526 SP FluorImager GE Healthcare 488 nm 530 DE 30 Storm GE Healthcare Blue fluorescence mode VDS CL GE Healthcare Transmission UV Low Ultracam Gel Imager Ultra Lum UV Yellow Filter 990 0804 07 Omega Systems Ultra Lum UV 520 nm Polaroid Camera Polaroid UV SYBR Safe Photographic Filter S27100 FOTO Analyst UV Fluorescent Green 1260 2034 Express Investigator Plus Luminary FOTODYNE FOTO Analyst Minivisionary FOTODYNE UV Fluorescent Green 62 4289 FOTO Analyst Apprentice FOTODYNE UV Fluorescent Green 1562 2535 FOTO Analyst Luminary FOTODYNE UV Fluorescent Green 12260 2056 FCR 10 Polaroid UV 83 4218 FUJI FLA 3000 FUJI Film 473 nm 520LP BioDocIt AC1 EC3 BioSpectrum UVP 302 nm SYBR Green 38 0219 01 or SYBR Gold 38 0221 01 Gel Logic Kodak UV 535 nm WB50 Syngene Instruments Syngene UV 500 600 nm Shortpass filter 125 Two Step Loading of E Gel Agarose Gels Introduction Recommended Volumes Loading Buffer 126 For optimal results follow the guidelines for preparing your DNA sample as described in this section The recommended total sample volume for each gel type is listed in the table below Note For best results keep all sample volumes uniform If you do not have enough samples to load all wells of the gel load an equal volume of deionized water all E Gel gels or buffer containing the same salt concentration as samples
93. iBase fits neatly on the Real time Transilluminator and power is provided through a shared power cord adapter included with the E Gel iBase Power System With the matching amber filter mounted on top of the iBase included with the E Gel Safe Imager Real time Transilluminator you can follow the migration of DNA bands while they are running or document your results at the end of the run directly iBase and Safe Imager Integrated System E Gel iBase Power System Amber Filter E Gel PowerBase E Gel The E Gel PowerBase Version 4 figure below is an easy to use automated PowerBase device specifically designed to simplify electrophoresis of single comb or double comb E Gel agarose gels from Invitrogen The E Gel PowerBase is a base and a power supply all in one device The operation of the E Gel PowerBase v 4 is controlled by two buttons on top of the base The left button is for a double comb run and right button is for a single comb run see the label on the unit To select different electrophoresis runs for the PowerBase do one of the following page 30 for details e Press and release the button run OR e Press and hold the button pre run electrical outlet B light adaptor pole buttons bem pole Top Bottom E Gel Base The E Gel Base see figure below previously available from Invitrogen connects to a power supply and is used for electrophoresis of E
94. iewing and documenting the gel or sample always switch the unit off 1 Place the Amber filter unit on top of the sample as shown below or use the viewing glasses when excising bands from DNA gels 2 Switch the E Gel Safe Imager Real time Transilluminator on using the ON OFF button in one of these ways e To turn on the light for 30 seconds press and release the ON OFF button The LED indicator light will be a flashing green throughout the run e To turn on the light for 5 minutes press and hold the ON OFF button for a few seconds The LED indicator light will turn a steady green followed by a flashing green the last 30 seconds of the run Any SYBR Safe stained DNA present should be immediately visible after light is on and amber filter unit or viewing glasses are in position 3 Toturn off the light press and release the ON OFF button The LED indicator light will turn red Note A flashing red LED indicates an error Wait until the LED turns a steady red before turning on the device again If the LED does not turn red after the run disconnect the Safe Imager and try again after a few minutes If this problem persists contact the Invitrogen Technical Service page 131 Continued on next page Safe Imager Blue Light Transilluminators Continued Safety Information Safe Imager Blue Light Transilluminator The Safe Imager Blue Light Transilluminator is an electrical device Never touch the power cord or outlet with wet h
95. into the collection well see next page amp invitrogen ee Retrieve DNA from bottom row Continued on next page 71 Running E Gel CloneWell and Collecting DNA Continued Running in Reverse Direction 78 Note The E Gel iBase Power System is pre programmed with a program to run E Gel agarose gels in a reverse direction 1 Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the REVERSE E Gel Program using the Up Down A V buttons to change the program If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons the maximal run time for reverse running is 3 minutes To start electrophoresis press the Go button a green light will illuminate to show that the run is in progress The LCD display will show the count down time while the run is in progress During this period of time monitor the run over a Safe Imager When you see the band of your interest migrating into the well press the Go button to stop the run and collect the DNA from the well using a pipette E Gel agarose gels can only be used once Do not re use them E Gel CloneWell gels already contain the SYBR Safe DNA gel stain and therefore do not have to be stained Visualizing E Gel CloneWell Agarose Gels Introduction Viewing E Gel CloneWell Agarose Gels Imaging E Gel Clon
96. irectly into the wells The E Gel agarose gel should be loaded within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples The proprietary fluorescent nucleic acid stain in E Gel SizeSelect agarose gels is more sensitive than ethidium bromide For downstream applications such as cloning or sequencing be sure to load enough DNA for your application as quantities of DNA that are sub optimal for your purposes can still produce a strong signal We recommend using the following DNA molecular weight markers for different types of E Gel SizeSelect agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel SizeSelect agarose gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng 25 bp DNA Ladder 10597 011 markers in a E Gel SizeSelect 2 agarose 84 volume of 5 10 ul 50 bp DNA Ladder 10416 014 100 bp DNA Ladder 15628 019 Low DNA Mass Ladder 10068 013 Run Time Estimation for SizeSelect Agarose Gels Introduction Refer to the Run Time Table below to estimate the run time for your DNA fragment to reach the reference line and then from the reference line to reach the collection well Be sure to monitor your gel during the run If the amount of DNA is low the band may not be visible Viewing the gel in a darkened room may improve visualization The Invitrogen 50 bp ladder see page ix can be run
97. ith multichannel pipettors or automated liquid handling systems e High Throughput E Gel Electrophoresis System is designed for electrophoresis of 96 DNA samples per gel This system is compatible for use with multichannel pipettors or automated liquid handling systems Gel Selection Choosing a Gel for Your Application Gel Type E Gel EX E Gel single comb with ethidium bromide E Gel with SYBR Safe E Gel CloneWell E Gel SizeSelect E Gel double comb with ethidium bromide E Gel 48 Gel E Gel 96 Gel To obtain the best results for your application it is important to choose the correct agarose percentage and well format The table below lists the various types of gel and resolution for each gel type No Sample Sample Resolution ih Loading Wells Volume eet mom 10 sample 1 marker 12 12 20 ul 8 sample 20 25 ul marker 8 sample 20 25 ul marker 16 sample 20 ul 10 ul 15 ul 15 ul 2 marker 48 sample 4 marker 96 sample 20 ul 20 ul 8 marker Wells compatible for loading with a multichannel pipettor Apparatus Compatibility Gel Type E Gel CloneWell E Gel with SYBR Safe E Gel EX E Gel SizeSelect E Gel single comb and double comb with ethidium bromide E Gel 48 96 Gel 100 bp 5 kb 50 bp 2 kb 800 bp 10 kb 100 bp 5 kb 100 bp 2 kb 20 bp 500 bp 100 bp 5 kb 100 bp 2 kb 100 bp 6 kb 50 bp 2 kb 1 kb 10 kb 10
98. l Load the recommended sample volume per well volume per well from the wells Use the Two Step Loading method page 126 Wells damaged during comb Remove the comb gently without damaging the wells removal Continued on next page Expired gel used 94 Troubleshooting Continued Problem High background suboptimal or no image Stripes visible on image Band of interest below collection well Low volume in collection well Low yield bands smeared Low yield bands not visible Cause No filters or wrong filter set Photographic settings not optimal No IR coating on camera when using an UV system Run time too long Well not refilled prior to collection Excess quantity of DNA Low quantity of DNA Solution Refer to page 123 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel SizeSelect agarose gels empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use Reverse program as described in the E Gel Technical Guide to run band back into collection well Fill the second row of wells with sterile water prior to running your band of interest into the wells Collect DNA from the well in two or more fractions refill with water after each collection Load the recommended amount
99. l 96 gel 20 ul Add deionized water to the required Add deionized water to the required amount of DNA to bring the total amount of DNA to bring the total sample volume to 15 ul sample volume to 20 ul Loading buffer is optional See page 30 for more details Continued on next page Sample Preparation for E Gel 48 96 Gels Continued DNA Molecular We recommend using the following DNA molecular weight markers for different Weight Markers types of E Gel agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel agarose gels containing ethidium bromide E Gel 48 gels 1 1 Kb Plus DNA Ladder 10787 018 Load 100 250 ng of TrackIt 1 Kb Plus DNA Ladder 10488 085 pa evo 15 ul in marker well Use 500 bp DNA Ladder 10594 018 a buffer containing the E Gel High Range DNA Marker 12352 019 same salt concentration 2 TrackIt 50 bp DNA Ladder 10488 043 ap Jour sampes TrackIt 100 bp DNA Ladder 10488 058 50 bp DNA Ladder 10416 014 100 bp DNA Ladder 15628 019 Low DNA Mass Ladder 10068 013 E Gel Low Range Quantitative DNA Marker 12373 031 4 Tracklt 25 bp DNA Ladder 10488 022 TrackIt 50 bp DNA Ladder 10488 043 25 bp DNA Ladder 10597 011 50 bp DNA Ladder 10416 014 E Gel Low Range Quantitative DNA Ladder 12373 031 E Gel 96 gels E Gel 96 High Range DNA Marker 12352 019 Load 100 250 ng of 2 E Gel Low Range Quantitative DNA
100. l See page 30 for more details One Step Loading Load samples in the appropriate sample volume directly into the wells The Method E Gel agarose gel should be loaded within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples Continued on next page 44 Sample Preparation for E Gel with SYBR Safe continued DNA Molecular Weight Markers E Gel 1 2 with SYBR Safe E Gel 2 with SYBR Safe We recommend using the following DNA molecular weight markers for different types of E Gel agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel with SYBR Safe agarose gels E Gel 1 Kb Plus DNA Ladder E Gel High Range DNA Marker 100 bp DNA Ladder 1 Kb Plus DNA Ladder High DNA Mass Ladder E Gel 1 Kb Plus DNA Ladder E Gel Low Range Quantitative DNA Marker 25 bp DNA Ladder 50 bp DNA Ladder 100 bp DNA Ladder Low DNA Mass Ladder 10488 090 12352 019 15628 019 10787 018 10496 016 10488 090 12373 031 10597 011 10416 014 15628 019 10068 013 Load 500 750 ng markers in a volume of 20 ul 45 Loading E Gel with SYBR Safe Gels Introduction Pre running E Gel with SYBR Safe Installing iBase Power System Alone Installing iBase and Safe Imager 46 After you have prepared your samples you are ready to proceed with electrophoresis Instructions are provided below
101. l keep your blotting apparatus ready before opening the gel The blades on the E Gel Opener are extremely sharp Do not insert your fingers into the area housing the blades Pick up the E Gel Opener by holding the large knob only see Figure 1 above Exercise caution when handling and cleaning the E Gel Opener Dispose of blades in a needle disposal container or a Sharps disposal box Continued on next page E Gel Opener Continued Procedure for The following section provides instructions to open an E Gel cassette Before Opening an E Gel beginning you should wear safety goggles and gloves Single Comb and 1 Double Comb Cassette Place the E Gel Opener on a flat surface with the knob extending off the edge of the laboratory bench and facing the user Set the E Gel Opener to its widest open position by turning the knob counterclockwise Insert the E Gel into the E Gel Opener so that two opposing sides of the gel cassette are aligned with the blades see Figure 1 Position the E Gel such that the two sides fit into the grooves housing the blades Turn the knob steadily clockwise to bring the blades in contact with the E Gel cassette As the knob is tightened you will hear a series of pops Continue to turn the knob until the resistance increases Stop turning the knob as soon as the E Gel cassette begins to lift off the surface of the platform Two sides of the E Gel will now be unsealed Note Once you observ
102. ld the time button to increase the time continuously To increase the run time while a run is in progress see next page To manually interrupt or stop a run see page 104 Each E Gel 48 and 96 gel is supplied individually wrapped and ready for use Use short rigid tips for robotic loading To load your samples on the E Holder refer to page 107 for detailed instructions 1 Open the package and remove the gel 2 Remove the plastic comb from the gel 3 Slide the gel into the two electrode connections on the Mother E Base or Daughter E Base The two copper electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base as shown below 4 When the gel is properly inserted into the base a fan in the base will begin to run and a red light will illuminate at the lower left corner of the base The digital display will show the appropriate time for a selected program or last time setting Ready Mode a Electrode _ Electrode Note If you accidentally inserted a gel into the base before selecting program EG remove the gel select program EG and then reinsert the gel in the base Continued on next page 101 Loading E Gel 48 and 96 Gels Continued Method of We recommend the following methods of sample loading based on the gel type Loading Samples Gel Type Method of Loading E Gel 48 Manual multichannel pipettor load samples into alternate wells of
103. lls Wells damaged during comb Remove the comb gently without damaging the wells removal Continued on next page 81 Troubleshooting Continued Problem Failure Mode indicated by a flashing red light and continuous rapid beeping Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency Band of interest below collection well Low volume for collection Low yield 82 Cause Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal No IR coating on camera Used a UV light source to visualize DNA Run time too long Missed refilling water Band is too big Solution Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page 123 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel CloneWell empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use
104. ls on UV transilluminators Although the viewing glasses do block UV light they are not designed for use as UV safety glasses Do not leave the Safe Imager switched on for extended periods of time After viewing and documenting the gel or sample always switch the unit off Do not attempt to open the Safe Imager Operating the Safe 1 Ensure that the Safe Imager Blue Light Transilluminator is placed on a Imager Blue Light Transilluminator level bench and there is enough air circulation around the unit to prevent overheating Plug the power cord into electrical outlet 2 Before handling your gel or sample ensure that the personal safety equipment you are using is appropriate for the hazards posed by the chemicals that may be present Place the gel or sample onto the surface of the Safe Imager transilluminator 3 Place the amber filter unit on top of the sample or stained gel If you are using a gel that is larger than the viewing area you may rest the amber filter unit directly on top of the gel or forgo the amber filter unit and rely solely on the viewing glasses The viewing glasses are also useful when excising bands from DNA gels as they allow the bands to be visualized while leaving the gel surface unobstructed Make sure to use either the Safe Imager amber filter unit or Safe Imager viewing glasses they not only help to visualize the SYBR stained DNA but also prevent prolonged exposure of your eyes to the intense bl
105. minutes If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons up to the maximal run time of 20 minutes To start electrophoresis press the Go button a green light illuminates to show that the run is in progress The LCD displays the count down time while the run is in progress The run will stop automatically when the programmed time has elapsed The iBase signals the end of the run with a flashing red light and rapid beeping for 30 seconds followed by a single beep every minute The LCD displays Run Complete Press Go Press and release the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program Remove the E Gel EX cassette from the iBase You are now ready to proceed to imaging or any other application with the gel Continued on next page 61 Running E Gel EX Agarose Gels Continued Interrupting a Run Using iBase Note 62 You can interrupt an electrophoresis run on the iBase at any time by pressing and releasing the Go button to stop the current The stopped current is indicated by a flashing red light and the digital display flashes to indicate that the run was interrupted The display also shows Press GO to Run Hold Go to Reset You can remove the gel from the iBase to check the progress of the run Then To continue the run from the point
106. mphocytes with or without S9 metabolic activation e Compared to ethidium bromide SYBR Safe DNA gel stain causes fewer mutations in the standard Ames test Weakly positive results occurred in only four out of seven Salmonella strains and only with activation by a mammalian S9 fraction e Asingle oral administration of SYBR Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5000 mg kg View studies documenting the safety of SYBR Safe in the SYBR Safe White Paper document available from http probes invitrogen com media publications 494 pdf SYBR Safe DNA gel stain is not classified as hazardous waste but as disposal regulations vary please contact your safety office or local municipality for appropriate SYBR Safe disposal in your community Continued on next page 19 SYBR Safe DNA Gel Stain Continued Spectrum of SYBR Bound to nucleic acids SYBR Safe stain has fluorescence excitation maxima at Safe Visualization of SYBR Safe Cloning Benefits of SYBR Safe 20 280 and 502 nm and an emission maximum at 530 nm see figure below Normalized fluorescence excitation and emission spectra of SYBR Safe DNA gel stain determined in the presence of DNA Excitation Emission Fluorescence excitation Fluorescence emission 300 400 500 600 700 Wavelength nm Detect DNA bands stained with SYBR Safe DNA gel stain using a blue light transilluminator a standard UV t
107. n steady red light and the digital display will flash to indicate that the run has been interrupted You can remove the gel from the mother or daughter base to check the progress of the run Then e Tocontinue the run from the point at which it was stopped reinsert the gel and press and release the power button e To cancel the rest of the interrupted run press and hold the power button for a few seconds The digital display will reset and the base will return to ready mode If desired you can then program a new run time In case of a power failure the run will continue when the power resumes The mother or daughter base will signal the end of the run as described on the previous page except the light will be an alternating red green and ER is displayed in the digital display to indicate that a power failure has occurred during the run 119 E Gel 96 Mother Daughter Base Quick Reference Guide Introduction 120 A quick reference guide for operating the E Gel 96 mother and daughter base is provided below Operating modes and electrophoresis runs are described below Mode Action Sound Light Digital EL Dase plugged in Ready with no current flowing through gel End of run Run ends after a power failure during the run Pause manually end the run Return to Ready mode after an automatic stop Mother base connected to an electrical outlet E Gel 96 cassette inserted into a base
108. nates on the base when the cassette is correctly base inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Poor resolution Sample is overloaded Do not load more than the recommended amount of smeared DNA sample per band see page 56 2 em High salt concentration Dilute your high salt samples as described on page 31 migration Aberrant pre run step Do not pre run E Gel SizeSelect agarose gels Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 1 minute of sample immediately after sample loading loading Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current time or high current during resulting in poor band migration or a melted gel Do the run not run the gel longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 25 minutes longer run times Sample leaking Sample is overloaded Load the Load the recommended sample volume per wel
109. nd allows tracking of your samples during photo documentation of the gel During electrophoresis samples migrate between the wells of the row below For example the bands of the lane B11 migrate between well C11 and C12 see figure on the next page This configuration provides a 1 6 cm run length allowing resolution between 100 bp and 10 kb The staggered well format of the gel cassette is compatible with automated liquid handling devices that use 8 12 or 96 tip loaders During sample loading the samples will fall onto the slopes of the wells and be drawn into the wells by capillary force Diagram of A diagram of the E Gel 96 cassette is shown below For details on the gel see E Gel 96 Cassette previous page didus o a 5 g 7 E 5 a 1i 12 M l Copper electrode li Lar 2 __ Sample migration Fir gt eb CB ot bt gt oe between wells i E Gel go Aes gra invi roget n T i eee HOPPED RIGGINS UH barcode 15 E Base Power Supply E Base Mother E Base 16 Two types of bases are available from Invitrogen The Mother E Base Invitrogen Cat no EB MO03 has an electrical plug that can be connected directly to an electrical outlet and is used for electrophoresis of one E Gel 48 E Gel 96 E PAGE 48 or E PAGE 96 gels available from Invitrogen The Daughter E Base Invitrogen Cat no EB D03 connects to the Mother E Base and together they can be used for the el
110. nt of sample to run on an E Gel SizeSelect agarose gel If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution E Gel SizeSelect 2 agarose gel Single DNA Multiple DNA Optimal Sample Maximum Band Bands Amount Sample Amount 1 300 ng 1 100 ng band 500 ng The recommended total sample volume for E Gel SizeSelect is 20 25 pl Load 5 10 ul DNA molecular weight marker into the small middle well lane M Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Prepare your samples as described below 1 Prepare the samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 25 ul 2 Prepare the DNA molecular weight marker by adding deionized water to the required amount of DNA to bring the total sample volume to 5 10 ul Continued on next page 83 Sample Preparation for E Gel SizeSelect Agarose Continued Loading Buffer Loading Method Note DNA Molecular Weight Markers Instead of water you may use a loading buffer to prepare samples or DNA molecular weight marker See page 30 for more details Tracking dye can be used but must be very dilute to avoid masking the bands Load samples in the appropriate sample volume d
111. ntinuously and automatically stops at 00 Selected program EP or EG Using E Holder Platform Introduction Procedure The E Holder Platform is designed to hold E Gel 48 and96 gels during robotic loading Use the E Holder when you need to load multiple gels on a robotic platform while other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run E Gel 96 gels To obtain the best results run E Gel 48 or96 gels on the Mother E Base or Daughter E Base within 15 minutes after loading on E Holder 1 Place the E Holder on the robotic platform 2 Open the package and remove the E Gel 48 or 96 gel 3 Remove comb from the E Gel cassette 4 Place the E Gel cassette in the E Holder Align the bottom left end of the cassette in the lower left alignment corner of the E Holder as shown in the figure below E Holder ae os oe ore es a L3 ES E ED E EJ ES eee ES EZ EJ EJ EE C6 CR DEC E ccr tcr t C C3 p S ku EB ES E3 p E rp EE iF rg aa aAa E3 3 E Gel 96 gel E r3 CE DER LEN DE Bes Dd LA LEA DEA DUE CUR DG DG D Ld LJ Ld c3 t3 GGG G rtt LL Lt LE E LD E Alignment corner 5 Setup your robotic system to load samples into the E Gel 48 or 96 gel placed on an E Holder Program your robotic system to load the samples approximately 5 minutes before the previous electrophoresis run is complete This
112. nue holding the Go button and insert the power plug into the electrical outlet Then connect the cable to the iBase unit Release the Go button and connect the iBase to the computer with a USB A to B cable A into the computer B end into the iBase The computer should now begin to search for the iBase This step may take several minutes A B The program indicates that it is searching for the iBase The program indicates that the iBase has been found Press Next to begin the iBase Firmware Update Do not disconnect or use device until iBase Update is complete Once the update is complete the program will indicate that the update was successful 10 Disconnect the USB cable from the iBase device 11 The iBase is now updated In case a message The Update Failed Retry the program and if the problem persists contact Technical Support see page 131 for further assistance 123 Parameters for E Gel iBase Programs Introduction The E Gel iBase Power System contains a number of different programs to run different types of E Gel agarose gels Refer to the table below for the run parameters default time and maximum allowable time for each program Program Gel Tvpes iii eee Default Run Maximal Run Number yp 5 Time min Time min 0 PRE RUN 2 1 E Gel 0 8 1 2 2 E Gel 0 8 2 0 40 2 E Gel 496 E Gel 4 40 3 E Gel double comb 0 8 2 E Gel DC 20 4 E Gel CloneWell 0 8
113. numbers are labeled with fluorescent dye that transfers to the image and allows tracking of your samples during photo documentation of the gel In addition each E Gel 48 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial barcode readers page 98 The separation range for E Gel 48 gels is listed below 196 E Gel 48 2 E Gel 48 100 bp 300 bp 300 bp 700 bp 700 bp 1200 bp 1200 bp 2000 bp 4 E Gel 48 5 bp 40 bp 40 bp 80 bp 80 bp 175 bp 175 bp 300 bp 300 bp 600 bp 13 High Throughput E Gel Electrophoresis System High Throughput The system consists of the following components E Gel Electrophoresis System E Gel 96 gels Each E Gel 96 gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format and is designed for high throughput agarose electrophoresis of nucleic acids E Base Electrophoresis Device The E Base is a base and a power supply all in one device and is an easy to use pre programmed device specifically designed for electrophoresis of E Gel 48 and 96 gels E Holder Platform The E Holder Platform is designed to hold E Gel 96 gels during robotic loading The E Holder is used to load multiple gels on a robotic platform while other gels are running on the E Base E Editor 2 02 Software The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and
114. of DNA Load the recommended amount of DNA View gel in darkened room or use 50 bp ladder as reference marker Refer to Run Time Table to determine when to collect the sample 95 Electrophoresis of E Gel 48 96 Gels Sample Preparation for E Gel 48 96 Gels Introduction Materials Needed Amount of DNA Total Sample Volume Preparing Samples Loading Buffer 96 E Gel 48 and E Gel 96 gels are designed for medium and high throughput electrophoresis of DNA fragments For optimal results follow the guidelines for preparing your DNA sample as described in this section e DNA sample e Loading buffer optional e Molecular weight markers page 97 Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng per lane for samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution The recommended total sample volume for each gel type is listed in the table below Note For best results keep all sample volumes uniform If you do not have enough samples to load all wells of the gel load an equal volume of buffer containing the same salt concentration as samples into any empty wells Gel Type Total Sample Volume E Gel 48 gel 15 ul E Gel 96 gel 20 ul Prepare your samples based on the loading method used as described below E Gel 48 gel E Ge
115. office for appropriate disposal methods Cleaning and After use clean the E Gel Opener with mild detergent and water to remove any Storage excess agarose ethidium bromide and plastic from the platform Use a squirt bottle and wipe the platform dry with a clean tissue Do not insert your fingers into the area housing the blades and do not immerse the E Gel Opener in water as the blades may rust Store the E Gel Opener at room temperature 10 Gel Knife Introduction Cleaning and Storage The Gel Knife is used to open the cassette for E Gel EX agarose gels See page 64 for details on usage Sharpened edge Clean the Gel Knife with mild detergent and water after use and store at room temperature 11 Medium Throughput E Gel Electrophoresis System Medium The system consists of the following components Th roughput e E Gel 48 gels Each E Gel 48 gel contains 48 sample lanes and 4 marker E Gel l lanes and is designed for medium throughput agarose electrophoresis of Electrophoresis nucleic acids System e E Base Electrophoresis Device The E Base is a base and a power supply all in one device and is an easy to use pre programmed device specifically designed for electrophoresis of E Gel 48 and 96 gels e E Editor 2 02 Software The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and documentation The E Editor 2 02 software can be
116. om Reference Line to Collection Well Retrieving DNA Once the band reaches the reference line refill the second row again with sterile water until the well is full some pre filled water is lost during the run Note If more concentrated DNA is desired do not completely fill the bottom well This will result in the retrieved DNA being more concentrated Press the Go button to run the gel for the time listed in the table below until the band enters the collection well During this period of time monitor the run over a Safe Imager At the end of this run you may see the band of your interest migrating into the well Note We recommend monitoring the run in a darkened room for optimal results Small DNA amounts and low molecular weight bands may be difficult to view inside the well 1 Band Size From Reference Line to Collection Well 200 bp 1 2 minutes 400 bp 1 2 minutes 800 bp 1 2 minutes 1000 bp 1 2 minutes 2000 bp 1 5 2 5 minutes 3000 bp 1 5 2 5 minutes 4000 bp 2 3 minutes 6000 bp 2 3 minutes Collect DNA from the well using a pipette Proceed to your application using the collected DNA without any further purification You may continue to collect more DNA bands from the same well be sure to fill more water into the second row well or from other wells If your band of interest overruns the collection well and re enters the gel use the REVERSE E Gel program of the iBase Power System to run the band backwards
117. on located on the lower right corner of the mother base see figure below and daughter base The red light will change to a green light while the run is in progress E Gel 96 mother base to electrical outlet electrode ME connection E Ge 96 gel tS va vag barcode ES electrode 2 connection sU E power s EP button 9g ot a An timer button a gt D E Gel 96 S mother base ES P T digital timer oreo display While the run is in progress you can add to the run time by pressing the time button To interrupt or stop a run in progress see next page The mother base will signal the end of the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute The digital display will show the elapsed time up to 19 minutes with a negative sign since the end of the run Press and release the power button to stop the beeping The light will turn to a steady red and the digital display will show the last time setting Remove the gel cassette from the mother base and daughter base You are now ready to capture an image of the gel Note The bands in the gel will diffuse within 20 minutes Continued on next page Using E Gel 96 Mother Daughter Base Continued Interrupting an You can interrupt an electrophoresis run at any time by pressing and releasing Electrophoresis the power button to stop the current The stopped current is indicated by a Ru
118. or consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 This product is provided under an agreement only for the use in staining nucleic acids in gels 2004 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use Windows
119. or best results run the gel within 15 minutes of sample immediately after sample loading loading If you cannot run the gel immediately after sample loading use the Two Step Loading method page 126 Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current time or high current during resulting in poor band migration or a melted gel Do the run not run the gel longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 40 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well from the wells Use Use the Two Step Loading method page 126 Two Step Use the Two Step Loading method page 126 method page 126 Wells damaged during comb Remove the comb gently without damaging the wells removal Continued on next page 54 Troubleshooting Continued Problem Failure Mode indicated by continuous rapid beeping and Cassette Missing Hold Go to Reset or a steady red light Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency Cause Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal
120. ot critical 1 Mix RNA sample with RNase free water such that the final volume is 20 ul 2 Do not heat Load the entire sample onto the E Gel EX 3 Run RNA using the E Gel EX program program 7 for 10 minutes The only denaturing agent that is compatible with the E Gel EX system is Formamide 50 95 Lower concentrations are also acceptable There are two methods for denaturing your RNA sample to run on an E Gel EX agarose gel Method 1 1 Mix RNA 250 ng 2 ug sample with formamide to 50 95 such that the final volume is 20 ul Heat samples at 65 C for 5 minutes to denature RNA Place samples on ice immediately after heating Load entire sample onto E Gel EX gae M Run RNA using the E Gel EX program program 7 for 10 minutes Method 2 1 Mix RNA 250 ng 2 ug sample with RNAse free water or loading buffer such that the final volume is 20 ul 2 Heat samples at 65 C for 5 minutes to denature RNA Using other denaturing agents like Glyoxal Formaldehyde or Urea results in very poor separation and band morphology on E Gel EX It is not recommended to run samples that were loaded with RNA loading buffer on the same gel as samples that are loaded with water Continued on next page Loading E Gel EX Agarose Gels Introduction After you have prepared your samples you are ready to proceed with electrophoresis Instructions are provided below to load E Gel EX agarose gels which come as single comb forma
121. page 2 E Gel iBase Power System and E Gel PowerBase v 4 The E Gel iBase Power System and PowerBase v 4 are a base and a power supply in one device These power systems connect directly to an electrical outlet using the adaptor supplied with the base page 6 E Gel Safe Imager Real time Transilluminator This transilluminator emits blue light and is specifically designed for use with SYBR Safe stained DNA gels run on the E Gel iBase Power System page 22 E Gel Opener The E Gel Opener is an implement specifically designed to open any E Gel single comb double comb or E Gel with SYBR Safe gel cassette page 9 Gel Knife The Gel Knife is used to open E Gel EX cassettes page 11 The Low Throughput E Gel Electrophoresis System consists of the following components E Gel CloneWell E Gel EX E Gel SizeSelect E Gel with SYBR Safe E Gel single comb and E Gel double comb pre cast agarose gels next page E Gel iBase Power System or E Gel PowerBase v 4 The E Gel iBase Power System and E Gel PowerBase v 4 are a base and a power supply in one device These power systems connect directly to an electrical outlet using the adaptor supplied with the base page 6 8 E Gel Safe Imager Real time Transilluminator specifically designed for use with E Gel EX E Gel SizeSelect and SYBR Safe stained DNA gels run on E Gel iBase Power System not suitable for viewing e
122. pport invitrogen com jpinfo invitrogen com eurotech invitrogen com MSDS MSDSs Material Safety Data Sheets are available at www invitrogen com msds Product The Certificate of Analysis provides detailed quality control and product Qualification Limited Warranty qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 10076 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order In
123. pty wells Running E Gel with SYBR Safe Gels Introduction After you have loaded your samples you are ready to proceed with electrophoresis Instructions are provided below to run E Gel with SYBR Safe which come as single comb gels using the E Gel iBase Power System or E Gel PowerBase v 4 For details on using the E Gel agarose gels with the E Gel Base see page 122 Electrophoresis 1 Using iBase Power System Toggle between program minutes and seconds on the iBase by pressing the Mode button until the program blinks Select the program RUN E Gel using the Up Down A V buttons to change the program Note The iBase Power System has the option of speed runs if you want quick results See below for more information Default run time for RUN E Gel is 26 minutes If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons up to the maximal run time of 40 minutes Remove the comb and load your samples Be sure to load molecular weight markers and add water to any empty wells To start electrophoresis press the Go button a green light illuminates to show that the run is in progress The LCD displays the count down time while the run is in progress The run will stop automatically when the programmed time has elapsed The iBase signals the end of the run with a flashing red light and rapid beeping for 30 seconds followed by a
124. ransilluminator or a laser based scanner For photographing gels a special filter may be required refer to page 51 for more information By using the SYBR Safe blue light for visualization DNA damage is dramatically reduced thus improving cloning efficiency For more information see the brochure http www invitrogen com content sfs brochures 713 01825 011203 EGel bro pdf or the article on SYBR Safe in Quest Vol 2 Issue 2 available July 2005 www invitrogen com Quest also available from Technical Support page 131 Proprietary Fluorescent Nucleic Acid Gel Stain Introduction Disposal of E Gel EX and E Gel SizeSelect Agarose gels Spectrum of Proprietary Fluorescent Nucleic Acid Gel Stain Visualization of Proprietary Fluorescent Nucleic Acid Gel Stain Cloning Benefits of Proprietary Fluorescent Nucleic Acid Gel Stain A proprietary nucleic acid stain has been specifically developed for E Gel EX and E Gel SizeSelect This gel stain has high sensitivity with detection down to 1 ng band of DNA In addition this proprietary fluorescent nucleic acid stain can be viewed by blue light transilluminator significantly reducing DNA damage that can reduce cloning efficiency Dispose of E Gel EX and E Gel SizeSelect agarose gels as hazardous waste in the same manner as ethidium bromide containing gels Contact your safety office or local municipality for appropriate disposal in your communit
125. rned during the warranty period This warranty does not apply if the product has been damaged by accident abuse misuse or misapplication or from ordinary wear and tear This warranty shall be limited to the replacement of defective products It is expressly agreed that this warranty will be in lieu of all warranties of fitness and in lieu of the warranty of merchantability General Information Purpose of the Guide Shipping and Storage The E Gel Technical Guide contains information about the E Gel pre cast agarose gels and is intended to supplement the Quick Reference Cards supplied with E Gel agarose gels Details for sample preparation and electrophoresis conditions are included in this guide To request the Quick Reference Card ORC or for additional information contact Technical Support page 131 or download the appropriate ORC from our Website at www invitrogen com All E Gel agarose gels are shipped at room temperature Store E Gel pre cast gels at room temperature Do not allow the temperature to drop below 4 C or rise above 40 C Gels are guaranteed to be stable for at least 2 to 6 months upon receipt e Standard and Clear gels are stable for at least 6 months e E Gel EX and E Gel SizeSelect are stable for at least 3 months e E Gel with SYBR Safe are stable for at least 2 months Please refer to the expiration date printed on the packaging of your E Gel agarose gel All electrophoresis bases are shippe
126. rose gel Before loading the gel make sure you have a blue light transilluminator set up for viewing the bands See page 79 for instructions You must first pre run the E Gel CloneWell agarose gel for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your DNA fragments Each E Gel cassette is supplied individually wrapped and ready for use To set up and use an E Gel CloneWell agarose gel using an iBase Power System follow the instructions below If using only the iBase Power System attach the power cord of the iBase to the power inlet and then to the electrical outlet Use only properly grounded AC outlets and cords If using the iBase Power System and Safe Imager Real time Transilluminator 1 Place the iBase directly onto the E Gel Safe Imager Real time Transilluminator so that the legs of the iBase fit directly into the grooves of the Safe Imager as shown in the image below 2 Plug the short electrical cord of the E Gel Safe Imager Real time Transilluminator a into the power inlet of the iBase b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager c and connect the power cord to the electrical socket To power outlet Continued on next page Loading E Gel CloneWell Agarose Gels Continued Pre running 1 E Gel CloneWell 2 Pre running 3 E Gel CloneWell Plug the E Gel
127. s Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel with SYBR Safe using a CCD camera or a laser based scanner For photographing gels refer to page 125 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel with SYBR Safe While yielding similar sensitivities to ethidium bromide SYBR Safe is somewhat dimmer yet with a lower background than ethidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary Results with E Gel 48 Gels 1 E Gel 48 Gel Results obtained using a 1 E Gel 48 gel is shown in the figure below The gel was electrophoresed for 23 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view the bands You may vary the amount of markers loaded on the gel to improve gel imaging 19 Agarose GP M1243 46 6 7 BB TOTS MISTI zt Td ELI FR ESO DUAE CUR TEGEU ake ee m MIU mE p tl E X P a T E Fm mu Ld nm UE The gel contains following samples Lane 1 24 M lower left lower right 2 3 22 23 32 33 36 37 40 41 4 5 20 21 30 31 42 43 6 7 18 19 28 29 44 45 8 9 12 13 16 17 26 27 46 47 10 11 14 15 34 35 38 39 25 48 M
128. s DNA Ladder E Gel High Range DNA Marker Kb Plus DNA Ladder 500 bp DNA Ladder High DNA Mass Ladder TrackIt 1 Kb Plus DNA Ladder E Gel 1 Kb Plus DNA Ladder E Gel High Range DNA Marker 100 bp DNA Ladder 1 Kb Plus DNA Ladder High DNA Mass Ladder Tracklt 100 bp DNA Ladder TrackIt 1 Kb Plus DNA Ladder E Gel 1 Kb Plus DNA Ladder E Gel Low Range Quantitative DNA Marker 25 bp DNA Ladder 50 bp DNA Ladder 100 bp DNA Ladder Low DNA Mass Ladder Tracklt 25 bp DNA Ladder Tracklt 50 bp DNA Ladder TrackIt 10 bp DNA Ladder TrackIt 25 bp DNA Ladder Tracklt 50 bp DNA Ladder TrackIt 1 Kb Plus DNA Ladder 10 bp DNA Ladder 25 bp DNA Ladder 50 bp DNA Ladder 10488 090 12352 019 10787 018 10594 018 10496 016 10488 085 10488 090 12352 019 15628 019 10787 018 10496 016 10488 058 10488 085 10488 090 12373 031 10597 011 10416 014 15628 019 10068 013 10488 022 10488 043 10488 019 10488 022 10488 043 10488 085 10821 014 10597 011 10416 014 Load 100 250 ng markers in a volume of 20 ul Continued on next page 33 Sample Preparation for E Gel continued Double comb E Gel gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng E Gel High Range DNA Marker 12352 019 DO ene 10 ul in marker well Low DNA Mass Ladder 10068 013 High DNA Mass Ladder 10496 016 E Gel 1 Kb Plus DNA Ladder 10488 090 E Gel Low Range Quantitative DNA Marker 12373 031 Tracklt 50 bp DNA Ladder 10488 043 Tr
129. s damaged during comb Be sure to remove the comb gently without removal damaging the wells Over run the gel Accidentally selected a Select EG if you are using E Gel 96 gels For E Gel or need more time different program 48 gels select EG and then manually change the time to run gel to 20 minutes If you accidentally selected a different program and are at the beginning of the run stop the run and select the desired program If you are well into the run check the gel to see where the loading dye is running Estimate the amount of time remaining and then manually stop the run Failure Mode Defective cassette Disconnect the base and replace gel cassette with a indicated by a fresh gel cassette steady red and Press and release the power button to return to continuous rapid Ready Mode beeping and aaa Cold cassette Use a room temperature cassette stored at room flashing ER on PLA i an E Base temperature Avoid storing gel cassettes at 4 C Improper operating Use the E Base at room temperature 20 25 C conditions Speckles visible Dust fluorescing in same Make sure gel is clean before imaging SYBR Safe gel wavelength as SYBR Safe High background No filters or wrong filter set Refer to page 123 to determine the optimal filter sets suboptimal or no to use or contact the instrument manufacturer for image SYBR advice Safe gel Photographic settings not Optimize settings of your
130. s using E Gel EX agarose gels 1 and 2 An example of DNA samples run on an E Gel EX 1 agarose gel is shown below Samples were loaded in a total volume of 20 ul and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec lt WOON A TF FP WN EHE 1 E Gel EX Agarose Gel T invitrogen V jd e Lane Sample E Gel 1 Kb Plus DNA Ladder 5 kb PCR product 2 kb PCR product 1 kb PCR product 500 bp PCR product E Gel High Range DNA Marker 500 bp PCR product 1 kb PCR product 2 kb PCR product 5 kb PCR product E Gel 1 Kb Plus DNA Ladder An example of DNA samples run on an E Gel EX 2 agarose gel is shown below Samples were loaded in a total volume of 20 ul and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec Lane M 1 2 3 s S 4 Mm wom C x 6 n 7 7 E Gel EX Agarose Gel 3 10 invitrogen Sample E Gel 1 Kb Plus DNA Ladder 500 bp PCR product 200 bp PCR product 100 bp PCR product 50 bp PCR product E Gel 1 Kb Plus DNA Ladder 50 bp PCR product 100 bp PCR product 200 bp PCR product 500 bp PCR product E Gel 1 Kb Plus D
131. samples that are in a high salt buffer refer to page 31 Continued on next page 56 Sample Preparation for E Gel EX Agarose Gels Continued Loading Buffer Loading Method DNA Molecular Weight Markers Loading buffer is optional See page 30 for more details Load samples in the appropriate sample volume directly into the wells The E Gel agarose gel should be loaded within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples We recommend using the following DNA molecular weight markers for different types of E Gel EX agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel EX agarose gels E Gel EX 1 agarose E Gel EX 2 agarose E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng E Gel High Range DNA Marker 12352 019 markers in a volume of 20 ul High DNA Mass Ladder 10496 016 E Gel 1 Kb Plus DNA Ladder 10488 090 E Gel Low Range Quantitative DNA Marker 12373 031 Low DNA Mass Ladder 10068 013 57 Preparing RNA Samples for E Gel EX Agarose Gels Introduction Non Denaturing Conditions Denaturing Agents Denaturing Conditions Important 58 E Gel EX agarose gels can be used to run RNA samples RNA can be run under denaturing or non denaturing conditions Use non denaturing conditions only when checking for RNA quality where accurately determining size is n
132. se You should hear a snap when it is in place The Invitrogen logo should be located at the bottom of the base close to the positive pole See the diagram below A steady red light illuminates when the E Gel gel is correctly inserted Ready Mode electrical outlet light adaptor pole buttons bes pole Top Bottom Press and hold either button until the red light turns to a flashing green light This indicates that the 2 minute pre run has started At the end of the pre run the current automatically shuts off The flashing green light changes to a flashing red light and the PowerBase beeps rapidly Press and release either button to stop the beeping you will hear only one beep The light changes from a flashing red light to a steady red light The E Gel with SYBR Safe agarose gels are designed for manual loading of samples All wells in the gel must contain sample or water Avoid introducing bubbles while loading as bubbles will cause bands to distort il Remove the comb from the E Gel with SYBR Safe gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Load 20 ul of sample per sample well page 44 for sample preparation Load 20 pl 500 700 ng of the appropriate molecular weight markers page 45 Load 20 ul of water into any remaining em
133. se the Daughter E Base without a Mother without Mother E Base E Base The Daughter E Base does not have an electrical plug to connect to an electrical outlet Copper contacts in the base are Make sure the copper contacts in the base are intact damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before cassette used the specified expiration date Gel cassette is not inserted Remove cassette and reinsert a red light illuminates at properly into a base the lower left corner of the base a fan in the base begins to run and digital display indicates time for a selected program or last time setting Ready Mode when the gel is properly inserted into the base Poor Sample is overloaded Do not load more than 20 100 ng of sample DNA per resolution or band Less DNA is required since E Gel agarose gels smearing of are thinner bands High salt concentration Dilute your high salt samples as described on page 31 Very low volumes of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load an equal volume of sample buffer containing the same salt concentration as your sample into the empty wells Gel was not electrophoresed For best results run the gel
134. seconds e An Up button marked A to select between programs on the display and increase running time e A Down button marked V to select between programs on the display and decrease running time A LED light is located in the middle of the four buttons which indicates the status of the iBase The gel cassette is inserted into the two electrode connections at the lower half of the iBase At the back the E Gel iBase Power System contains a USB port and a power inlet The supplied power cord has a matching connector that inserts into the power inlet and connects the E Gel iBase Power System to the electrical outlet A separate stand alone power supply is not required to run the iBase The supplied USB cable can be connected to any internet ready computer to download firmware upgrades from the Invitrogen website see page 123 E Gel iBase Power System top view To Electrical Outlet LCD Display i i r1 i l Down 4 0 9 9 Mode button EE buton DE TED tight E Gel iBase Power System back view y AP I Power USB inlet port Continued on next page E Gel iBase iBase and Safe Imager Integrated System E Gel Safe Imager Real time Transilluminator Power System Continued E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator form an integrated system for running and viewing SYBR Safe stained E gels The
135. see page 22 Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the program Run CloneWell using the Up Down A V buttons Toggle between program minutes and seconds by pressing the Mode button until the minutes blink Enter the estimated run time to the Reference line see previous page using the Up Down A V buttons Press the Go button on the iBase to run your band of interest to reach the printed reference line just above the bottom row of wells The red light turns to a green light indicating the start of the run Monitor your gel occasionally during the run If your band of interest reaches the reference line press the Go button to stop the run Continue with the next section At the end of the run the iBase stops after the entered run time and displays a flashing red light and beeps rapidly If your band did not reach the reference line run the gel for a few more minutes until the band reaches the line Place the iBase and CloneWell on the Safe Imager to allow for monitoring during the run Run the program Run CloneWell with the estimated run time Reference Line Reference Line b lad f 4 VT Ti Bands reaching the Reference Line Run the gel until the band of interest reaches the Reference Line Continued on next page Running E Gel CloneWell and Collecting DNA Continued Electrophoresis of 1 Bands fr
136. set You can remove the gel from the iBase to check the progress of the run Then To continue the run from the point at which it was stopped reinsert the gel and press and release the Go button The light changes to steady green and the LCD display shows the count down time It is also possible to change the remaining run time but not the program as described on the previous page before continuing the run To cancel the rest of the interrupted run press and hold the Go button for a few seconds The LCD display will reset and the base will return to Ready Mode If desired you can then select a new program or run time as described on the previous page and rerun the gel Running in The iBase is pre programmed with a program to run E Gel agarose gels in the Reverse Direction reverse direction This is particularly useful for isolating fragments using E Gel Using iBase CloneWell and SizeSelect agarose gels 1 Toggle between program minutes and seconds by pressing the Mode button M until the program blinks 2 Select the REVERSE E Gel Program using the Up Down A V buttons to change the program 3 If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons the maximal run time for reverse running is 3 minutes 4 To start electrophoresis press the Go button a green light will illuminate to show that the run is in progress The LCD display
137. sette to be opened with a Gel Knife Invitrogen Cat no EI9010 for excision of DNA fragments or for blotting Procedure The following section provides instructions to open an E Gel EX cassette Before beginning put on safety goggles and gloves 1 Place the E Gel EX cassette on a flat surface with the wells facing upward 2 Insert the sharp edge of the gel knife a into the groove around the edge of the cassette edge b and lever the knife up and down c 3 Work around the perimeter of the entire cassette and repeat this action for every edge Continued on next page 64 Opening an E Gel EX Agarose Gel Cassette continued 4 The 4 sides of the cassette should be unsealed Lift off the top of the gel cassette 5 If you plan to transfer DNA from the gel by blotting only the main running gel is required Remove the upper and lower IEM see page 5 and well areas with the Gel Knife 6 If you plan to purify DNA from the gel excise the gel fragment Transfer the gel slice to a microcentrifuge tube Disposal of E Gel E Gel EX agarose gels should be disposed of as hazardous waste in the same EX Agarose Gels manner as ethidium bromide containing gels Contact your safety office or local municipality for appropriate disposal in your community 65 Results using E Gel EX Agarose Gels Introduction E Gel EX 1 Agarose Gel E Gel EX 2 Agarose Gel 66 On this page we display typical result
138. sions 200 x 110 x 43 mm Amber filter dimensions 121 x 138 x 31 mm Weight of Safe Imager 243g Weight of Filter 55g Electrical Requirements 48 VDC 0 8 A max Temperature Ambient 5 C to 40 C Built in Features LED light LED life 50 000 hours LED Specifications Array of 12 high power LEDs emitting at 480 5 nm The LEDs used radiate less than 10 Lumens each at 200 mA Included accessories Amber filter unit and viewing glasses for viewing results Adapter Specifications Use only the UL Listed adapter supplied with the starter kit or with the E Gel iBase Power System Input 100 240 VAC 50 60Hz 1A Output 48 VDC 0 8 A Continued on next page Product Specifications Continued E Gel PowerBase v 4 Specifications E Gel iBase Adaptor Specifications E Gel PowerBase Adaptor Specifications E Gel Base Specifications The specifications for E Gel PowerBase V 4 are listed below Dimensions 12 5 cm x 13cm x 13 5 cm Weight 1 19 Ibs 540 g with adaptor Safety UL listed and CE certified Temperature Ambient 15 C to 40 C Built in Features Alarm light LED The E Gel iBase is designed for use with an adaptor included with the iBase Use only the UL Listed original adapter supplied Input 100 240 V AC 50 60Hz 1A Output 48 VDC 0 8 A The E Gel PowerBase v 4 is designed for use with an adaptor included with the PowerBase Use only UL Listed Class 2 Direct Plug in Ad
139. splay shows the count down time It is also possible to change the remaining run time but not the program as described on the previous page before continuing the run e To cancel the rest of the interrupted run press and hold the Go button for a few seconds The LCD display will reset and the base will return to Ready Mode If desired you can then select a new program or run time as described on the previous page and rerun the gel The E Gel iBase is pre programmed with a program to run E Gel gels in a reverse direction This is particularly useful for isolating fragments using E Gel CloneWell and E Gel SizeSelect agarose gels 1 Toggle between program minutes and seconds by pressing the Mode button M until the program blinks 2 Select the REVERSE E Gel Program using the Up Down A V buttons to change the program 3 If you want to change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons the maximal run time for reverse running is 3 minutes 4 To start electrophoresis press the Go button a green light will illuminate to show that the run is in progress The LCD display will show the count down time while the run is in progress 5 TheiBase will signal the end of the run with a flashing red light and rapid beeping for 30 seconds followed by a single beep every minute while the LCD display will read Run Complete Press GO 6 Pressand releas
140. ss of resolution on E Gel agarose gels To obtain the best results dilute samples which contain high salt levels 2 to 20 fold 1 Take the volume listed below for the type of sample you wish to dilute Source Sample Volume Restriction Digest fragment size gt 1 kb 1 ul Restriction Digest fragment size lt 1 kb 5 10 ul PCR 1 5 ul Digest of 500 ng 1 ug DNA in 20 ul PCR reaction size of 50 ul 2 Dilute samples as described below for the type of gel you are using Gel Type Dilution E Gel CloneWell agarose Dilute samples with loading gel buffer deionized water or TE to a final volume of 20 25 ul E Gel single comb gel E Gel double comb gel E Gel with SYBR Safe E Gel EX gel E Gel SizeSelect gel E Gel 96 gel E Gel 48 gel Dilute samples with loading buffer deionized water or TE to a final volume of 15 ul Dilute samples with loading buffer deionized water or TE to a final volume of 20 ul 3l Electrophoresis of E Gel Agarose Gels Sample Preparation for E Gel Agarose Gels Introduction Materials Needed Amount of DNA Total Sample Volume Preparing Samples Loading Buffer One Step Loading Method 32 For optimal results follow the guidelines for preparing your DNA sample as described in this section e DNA sample e Loading buffer optional e Molecular weight markers page 33 Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng
141. ss Go 6 Press and release the Go button to stop the beeping The light turns to a steady red light and the LCD display shows the last selected time and program 7 Remove the E Gel cassette from the E Gel iBase You are now ready to proceed to imaging or any other application with the gel The E Gel iBASE is pre programmed with a program for quick runs to get a yes no result The program SPEED E Gel utilizes high power and is suitable for 0 8 1 2 and 2 E Gels only This program is limited to 7 minutes where the bands migrate less than half the length of the gel A run exceeding 7 minutes under these conditions results in a defective run This mode is not compatible with E Gel 4 gels Continued on next page 37 Running Single Comb and Double Comb Gels continued Interrupting a Run Using iBase Running in Reverse Direction Using iBase 38 You can interrupt an electrophoresis run on the E Gel iBase at any time by pressing and releasing the Go button to stop the current The stopped current is indicated by a flashing red light and the digital display flashes to indicate that the run was interrupted The display also shows Press GO to Run Hold Go to Reset You can remove the gel from the E Gel iBase to check the progress of the run Then e Tocontinue the run from the point at which it was stopped reinsert the gel and press and release the Go button The light changes to steady green and the LCD di
142. t 50 bp DNA Ladder 0 4 ug well Results with E Gel 48 Gels Continued 4 E Gel 48 Gel Results obtained using a 4 E Gel 48 gel is shown in the figure below The gel was electrophoresed for 20 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view the bands You may vary the amount of markers loaded on the gel to improve gel imaging 496 Agarose HR M1234567 8 9 1011 1213 14 15 16 17 18 19 20 21 22 23 24 M To ras Sars ae ee d b f Lo Ld i The gel contains following samples Lane Sample 46 47 M upper left 10 bp DNA Ladder 1 ug well 1 20 21 38 39 48 M lower right 25 bp DNA Ladder 0 5 ug well 24 25 M lower left 50 bp DNA Ladder 0 5 ug well 26 27 M upper right 100 bp DNA Ladder 0 5 ug well 8 13 14 34 35 E Gel Low Range Quantitative DNA Ladder 10 ul well 2 9 Synthetic 21 mer siRNA short interfering RNA 100 ng well 6 7 dsRNA diced cut with Dicer enzyme 100 ng well 4 5 Undiced dsRNA 100 ng well 22 ssDNA 60 mer 200 ng well 23 ssDNA lane 22 annealed to form dsDNA 60 bp 100 ng well 9 10 44 45 PCR product Hinf I cut 11 12 36 37 PCR product Aat II cut 15 28 29 PCR product 40 bp 100 ng well 16 30 31 PCR product 72 bp 100 ng well 17 32 39 PCR product 150 bp 100 ng well 18 40 41 PCR product 240 bp 100 ng w
143. t using the E Gel iBase Installing iBase If using the iBase Power System without an E Gel Safe Imager Connect the Power System cord with the transformer to the power inlet of the iBase and plug the other Alone end of the power cord into an electrical outlet Use only properly grounded AC outlets and cords Installing iBase If using the iBase Power System and Safe Imager Real time Transilluminator and Safe Imager 1 Place the iBase directly onto the E Gel Safe Imager Real time Transilluminator so that the legs of the iBase fit directly into the grooves of the Safe Imager as shown in the image below 2 Plug the short electrical cord of the E Gel Safe Imager Real time Transilluminator a into the power inlet of the iBase b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager c and connect the power cord to the electrical socket To power outlet Continued on next page 59 Loading E Gel EX Agarose Gels Continued Inserting the E Gel EX Agarose Gel Method of Loading Samples Loading E Gel 60 1 Open the package and remove the gel Gently remove the comb from the E Gel EX agarose gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Slide the cassette into the two electrode conn
144. t will illuminate a steady red to show that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the program PRE RUN 2 minutes using the Up Down A V buttons to change the program Press the Go button to pre run the gel The LED light changes to green light to indicate that the cassette is in the pre run mode After two minutes pre run stops automatically as indicated by a red light and a beeping sound Continued on next page 35 Loading Single Comb and Double Comb Gels continued Pre running Using PowerBase v 4 Method of Loading Samples One Step Loading Method 36 1 Plug the PowerBase v 4 into an electrical outlet using the adaptor plug 2 Open the package containing the gel and insert the gel with the comb in place into the apparatus right edge first Press firmly at the top and bottom to seat the gel in the base You should hear a snap when it is in place The Invitrogen logo should be located at the bottom of the base close to the positive pole See the diagram below A steady red light will illuminate when the E Gel gel is correctly inserted Ready Mode electrical outlet light adaptor pole buttons ae pole 3 Top Bottom 4 Press and hold either button until the red light turns to a flashing green light This indicates th
145. t transilluminator such as the Safe Imager Blue Light Transilluminator or E Gel Safe Imager Real time Transilluminator see page x Refer to page 79 for instructions on using the Safe Imager Blue Light Transilluminator and E Gel Safe Imager Real time Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel CloneWell Note Do not use a UV transilluminator since UV light sources could lead to reduced cloning efficiencies Estimated Run Refer to the Run Time table below to estimate run times of your fragments to the Time to Reference Reference Line Same bands in different wells may migrate differently DNA Line fragment sizes amounts and salt content may also slightly affect the migration rates The run times indicated are estimates monitor your gel occasionally during the run Band Size Run time to Reference Line 200 bp 14 18 minutes 400 bp 15 19 minutes 800 bp 17 21 minutes 1000 bp 19 23 minutes 2000 bp 21 25 minutes 3000 bp 24 28 minutes 4000 bp 28 32 minutes 6000 bp 32 36 minutes Continued on next page 75 Running E Gel CloneWell and Collecting DNA Continued Electrophoresis of 4 Bands to Reference Line 76 If you haven t already done so place the E Gel iBase Power System over a blue light transilluminator Use the orange cover or orange goggles for viewing the bands For instructions using the Safe Imager Transilluminator
146. the two electrode connections on the base The LED light will illuminate a steady red to show that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure IT ann na I ae nam rogi et 4 Mr Continued on next page 87 Loading E Gel SizeSelect Agarose Gels Continued Loading E Gel All wells in the gel must be loaded with either sample or water Avoid SizeSelect introducing bubbles while loading as bubbles will cause bands to distort Agarose Gels Important Do not pre run E Gel SizeSelect agarose gel 1 Load 20 25 ul of sample into each well of the upper row page 83 for sample preparation 2 Load 5 10 ul 100 250 ng of the appropriately diluted molecular weight markers page 84 into the middle well lane M 3 Load 25 ul of deionized water into any remaining empty wells of the upper row 4 Load 25 ul of deionized water into all of the wells in the lower row collection wells Load 5 10 ul of deionized water into lane M of the lower row Load samples Load water in in wells 1 8 lt lt any empty wells Load DNA ladder into lane M Load water in all wells F Gel SizeSelect invitrogen 88 Running E Gel SizeSelect Agarose Gels Introduction Electrophoresis Using iBase Power System After you have loaded your samples you are ready to proceed with electrophoresis Instructions are provided below to run E Gel SizeSelect
147. the use of the strong mutagen ethidium bromide and reduces UV exposure e Maximize cloning efficiency since E Gel with SYBR Safe dramatically reduces DNA damage if using blue light transilluminators For details on SYBR Safe DNA gel stain see page 18 E Gel CloneWell agarose gels provide a simple method of DNA recovery in which the purified DNA is removed directly from the gel with a pipette In addition to the reasons stated above for E Gel With SYBR Safe use E Gel CloneWell 0 8 with SYBR Safe for e Fast and easy to purification of DNA fragments 100 bp 6 000 bp in size e Compatibility with blue light transillumination to dramatically reduce DNA damage E Gel SizeSelect 2 pre cast agarose gels contain a proprietary fluorescent nucleic acid stain allowing e Detection to 1 ng band of DNA e Fast and easy to purification of DNA fragments 50 bp 2 000 bp in size e Compatibility with blue light transillumination to dramatically reduce DNA damage Low Throughput E Gel Electrophoresis System Low Throughput E Gel Electrophoresis System System Components Applications The following components are available for low throughput electrophoresis E Gel CloneWell E Gel with SYBR Safe E Gel EX E Gel SizeSelect E Gel single comb and E Gel double comb pre cast agarose gels Gels are available in a variety of percentages Choose an appropriate gel based on your application see table on
148. thidium bromide stained gels page 23 E Gel Opener page 9 Note The E Gel Base previously available from Invitrogen can be used for electrophoresis of E Gel with SYBR Safe E Gel single comb and double comb agarose gels page 122 E Gel agarose gels are suitable for analyzing or purifying PCR products Restriction digests RT PCR reactions Low Throughput E Gel Well Formats E Gel Single Comb and Double Comb Gels Features of E Gel CloneWell and SizeSelect Agarose Gels The E Gel single comb and double comb gels are bufferless gels containing electrodes embedded in the agarose matrix Each gel contains an ion generating system TAE buffer system a pH balancing system and ethidium bromide for DNA staining and is packaged inside an UV transparent cassette To create a patented bufferless system each E Gel single comb and double comb cassette contains two ion exchange matrices IEMs that are in contact with the gel and electrodes The IEMs supply a continuous flow of ions through out the gel resulting in a sustained electric field required for running the gel see figure below See page 27 for product specifications Running gel Cathode Lower IEM Upper IEM The lower IEM near the The upper IEM near Upper IEM Anode anode contains Tris cations the cathode contains and ethidium bromide acetate anions OH AC Lower IEM Cu Trist E Gel CloneWell and E Gel Size
149. thidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary 79 m TM Results using E Gel CloneWell Agarose Gels Introduction On this page we display typical results using E Gel CloneWell Agarose Gels Example of E Gel An example of DNA samples run on an E Gel CloneWell is shown below CloneWell Bands that have entered the Collection Well completely are indicated by arrows The gel was visualized on a Safe Imager Transilluminator Bands that have entered the Collection Well CloneWell E Gel CloneWell was tested in cloning experiments using restriction based Cloning cloning Topo cloning and Gateway cloning In all cases colony counts were several fold higher using E Gel CloneWell compared to ethidium bromide gels Exact numbers may vary depending on the experiment but you should get many more colonies when using E Gel CloneWell for your fragment isolation 80 Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel CloneWell agarose gels No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before cassette the specified expiration date E Gel CloneWell cassette is Remove cassette and reinsert a steady red light not inserted properly intoa
150. tion of the E Gel iBase C Power System and E Gel Safe Imager Real time Transilluminator are subject to the following conditions UL us e Indoor use e Altitude below 2 000 meters LISTED E189045 e Temperature range 5 to 40 C e Maximum relative humidity 80 FE e Installation categories over voltage categories II Pollution degree 2 EN60825 1 e Mains plug is a disconnect device and must be easily accessible e Do not attempt to open the iBase or Safe Imager device To honor the warranty iBase and Safe Imager device can only be opened and serviced by Invitrogen e The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Invitrogen e The device must be connected to a mains socket outlet with protective earthing connections e Ventilation requirements no special requirements The E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator comply with part 15 of the FCC rules Operation of the devices are subject to the following conditions e The device may not cause harmful interference e The device must accept any interference received including interference that may cause undesired operation Ethrog Biotechnologies Ltd an Invitrogen company is the manufacturer and owner of the UL file For more information contact Technical Service page 131 or Ethrog Ethrog Biotechnologies Ltd Ness Ziona Science Park Bldg 22 P
151. to load E Gel with SYBR Safe available as single comb gels using the E Gel iBase or PowerBase v 4 For details on using the E Gel agarose gels with the E Gel Base see page 122 You must first pre run the E Gel with SYBR Safe agarose gel for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your DNA fragments Each E Gel cassette is supplied individually wrapped and ready for use To set up and use an E Gel with SYBR Safe gel using an iBase Power System follow the instructions below If you are using a PowerBase v 4 see next page If using only the iBase Power System attach the power cord of the iBase to the power inlet and then to the electrical outlet Use only properly grounded AC outlets and cords If using the iBase Power System and Safe Imager Real time Transilluminator 1 Place the iBase directly onto the E Gel Safe Imager Real time Transilluminator so that the legs of the iBase fit directly into the grooves of the Safe Imager as shown in the image below 2 Plug the short electrical cord of the E Gel Safe Imager Real time Transilluminator a into the power inlet of the iBase b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager c and connect the power cord to the electrical socket To power outlet Continued on next page Loading E Gel with SYBR Safe Gels continued
152. uction E Gel with SYBR Safe agarose gels contain the safer and environmentally friendly SYBR Safe DNA gel stain enabling visualization of bands with a blue light transilluminator thus minimizing DNA damage For optimal results follow the guidelines for preparing your DNA sample as described in this section Note For instructions to run E Gel 96 with SYBR Safe gels refer to the chapter Electrophoresis of E Gel 48 96 Gels page 96 Materials Needed DNA sample e Loading buffer optional e Molecular weight markers page 45 Amount of DNA Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng per lane E Gel 1 2 with SYBR Safe or 700 ng per lane E Gel 2 with SYBR Safe of samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution Total Sample The recommended total sample volume for E Gel with SYBR Safe is 20 pl Volume Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Preparing Prepare your samples by adding deionized water to the required amount of Samples DNA to bring the total sample volume to 20 ul For samples that are in a high salt buffer refer to page 31 Loading Buffer Loading buffer is optiona
153. ue light 4 Switch the Safe Imager transilluminator ON using the ON OFF switch located at the front of the instrument Any SYBR stained DNA present in solution or in gel bands should be immediately visible after the light is on and the amber filter unit or viewing glasses are in position Continued on next page 25 Safe Imager Blue Light Transilluminators Continued Imaging Cleaning and Maintenance 26 e Todocument your results you may use any standard imaging device Due to the small footprint the Safe Imager transilluminators may fit inside the cabinet of your current gel documentation system In many cases satisfactory results are obtained by placing the amber filter unit on top of the gel and photographing imaging using standard procedures e Your CCD documentation systems may already include an appropriate filter for imaging the gel see page 51 for filter guidelines and contact the manufacturer for filter specifications You may use this filter in place of the amber filter unit e The Safe Imager transilluminators have a very slim design compared to UV transilluminators the distance between the camera and the gel may have to be adjusted e After viewing or documenting the results switch the Safe Imager transilluminator off Clean the Safe Imager transilluminators with a dry cloth or with water and mild soap Ethanol may also be used Avoid damaging or scratching the glass surface of the Safe Imager
154. uired amount of DNA to bring the total sample volume to 5 10 ul Instead of water you may use a loading buffer to prepare samples or DNA molecular weight marker See page 30 for more details Do not use a tracking dye to avoid masking the bands For samples that are in a high salt buffer refer to page 31 Load samples in the appropriate sample volume directly into the wells The E Gel agarose gel should be loaded within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples Continued on next page Sample Preparation for E Gel CloneWell Agarose Gels Continued DNA Molecular We recommend using the following DNA molecular weight markers for E Gel Weight Markers CloneWell 0 8 with SYBR Safe to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel CloneWell 0 8 with SYBR Safe agarose gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 500 700 ng E Gel High Range DNA Marker 12352 0 0 eke Ina volume of 5 1 Kb Plus DNA Ladder 10787 018 10 ul High DNA Mass Ladder 10496 016 71 Loading E Gel CloneWell Agarose Gels Introduction Important Pre running E Gel CloneWell Installing iBase Power System Alone Installing iBase and Safe Imager 72 After you have prepared your samples you are ready to proceed with electrophoresis Instructions are provided below to load an E Gel CloneWell aga
155. um Bromide 18 Pak 18 gels G6018 08 E Gel 2 double comb with Ethidium Bromide 18 Pak 18 gels G6018 02 E Gel 48 Gels E Gel 48 1 with Ethidium Bromide gels 8 gels G8008 01 E Gel 48 2 with Ethidium Bromide gels 8 gels G8008 02 E Gel 48 4 with Ethidium Bromide gels 8 gels G8008 04 E Gel 96 Gels E Gel 96 2 with SYBR Safe gels 8 gels G7208 02 E Gel 96 1 with Ethidium Bromide gels 8 gels G7008 01 E Gel 96 2 with Ethidium Bromide gels 8 gels G7008 02 Continued on next page Accessory Products Continued Electrophoresis Bases DNA Molecular Weight Markers E Gel iBase USB Mini Cable E Holder Gel Knife E Gel Opener E Editor 2 02 Software The following electrophoresis bases are available from Invitrogen for electrophoresis of E Gel agarose gels e The E Gel iBase Power System Invitrogen Cat nos G6400 G6400EU G6400UK is used for electrophoresis of E Gel CloneWell E Gel EX E Gel SizeSelect single comb and double comb gels e The E Gel PowerBase v 4 available only in starter kits is used for electrophoresis of E Gel single comb and double comb gels e The Mother E Base Invitrogen Cat no EB M03 is used for electrophoresis of one E Gel 48 or 96 gel e The Daughter E Base Invitrogen Cat no EB D03 attaches to the Mother E Base and is used for electrophoresis of two or more E Gel 48 or 96 gels A large variety of DNA molecular weight markers for use with
156. upper right upper left eyre mem mE M i s ng EILDIZ High Mass DNA Ladder 4 ul well PCR product 317 bp 100 ng well PCR product 1 kb 100 ng well PCR product 3 kb 100 ng well PCR product 9 kb 100 ng well E Gel High Range DNA Ladder 10 ul well 1 Kb Plus DNA Ladder 0 5 ug well Continued on next page 109 Results with E Gel 48 Gels Continued 2 E Gel 48 Gel 110 Results obtained using a 2 E Gel 48 gel is shown in the figure below The gel was electrophoresed for 20 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view the bands You may vary the amount of markers loaded on the gel to improve gel imaging 2 Agarose GP i M123 4 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 M LEO I CT Qs LO OS LL OL OL LOL a n The gel contains following samples Lane Sample 1 24 M lower left lower right 100 bp DNA Ladder 0 4 ug well 2 3 22 23 34 35 38 39 PCR product 150 bp 100 ng well 4 5 8 9 20 21 40 41 PCR product 240 bp 100 ng well 6 7 17 18 30 31 41 42 PCR product 317 bp 100 ng well 8 9 16 17 28 29 44 45 PCR product 1 kb 100 ng well 10 11 14 15 26 27 45 47 PCR product 3 kb 100 ng well 12 13 36 37 E Gel 96 Low Range Quantitative Ladder 10 ul well 25 48 M upper left upper righ
157. vided by the equipment may be impaired if the equipment is used in a manner not specified by Invitrogen Ethrog Biotechnologies Ltd an Invitrogen company is the manufacturer and owner of the UL file For more information contact Ethrog Biotechnologies Ltd Ness Ziona Science Park Bldg 14 P O Box 444 Ness Ziona Israel 74103 documentation Y The Caution symbol denotes a risk of safety hazard Refer to accompanying Caution qms WEEE Waste Electrical and Electronic Equipment symbol LAJ 8 ENEN WEEE I Class II product Double Insulation Continued on next page 128 Explanation of Symbols and Warnings Continued E Gel iBase The E Gel iBase Power System complies with the Underwriters Laboratories Power System Inc regulation and the European Community Safety requirements Operation of the E Gel iBase Power System is subject to the following conditions e Indoor use C C e Altitude below 2 000 meters e Temperature range 5 to 40 C UL us e Maximum relative humidity 80 LISTED e Installation categories over voltage categories H Pollution degree 2 E189045 e Mains plug is a disconnect device and must be easily accessible e Do not attempt to open the iBase Device To honor the warranty iBase FRE device can only be opened and serviced by Invitrogen e The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Invitrogen e The device must be
158. vitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 131 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology 132 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Comm
159. will ensure that the loaded gel from the E Holder will be placed onto an E Base within the recommended time of 15 minutes 107 Visualizing E Gel 96 with SYBR Safe Agarose Gels Introduction Viewing E Gel with SYBR Safe Imaging E Gel with SYBR Safe Required Filters Important Exposure Time and Gain Setting 108 Bound to nucleic acids SYBR Safe DNA gel stain has fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate see below View E Gel with SYBR Safe using these instruments e Blue light transilluminator The Safe Imager Blue Light Transilluminator Invitrogen Cat no S37102 is designed specifically for use with SYBR Safe stained DNA gels Refer page 22 for instructions on using the Safe Imager Blue Light Transilluminator Blue light transilluminators available from on manufacturers are also compatible for use with E Gel with SYBR Safe e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources in combination with SYBR Safe stain could lead to reduced cloning efficiencie
160. within 15 minutes of immediately after sample sample loading loading If you cannot run the gel immediately after sample loading use the Two Step Loading method page 126 If you are using the E Holder program your robotic system to load the gel 5 minutes before the end of the previous gel s run A1 tip not aligned Be sure to align the Al tip properly prior to loading your samples on an E Gel 96 gel page 99 Expired gel used Use properly stored gels before the specified expiration date Longer electrophoresis run Longer run times cause an increase in the current time or high current during the resulting in poor band migration Do not run the gel run longer than the recommended time for each gel type Continued on next page 116 Troubleshooting Continued Uneven run on Differential salt concentration Be sure to load 15 ul of sample buffer containing the E Gel 48 gels in adjacent lanes same salt concentration as the sample into any remaining empty wells Keep all sample volumes uniform Slanted bands in Differential salt concentrations Prepare the marker in a buffer containing the same marker lanes on in adjacent lanes salt concentration as the samples E Gel 48 gels Sample leaking Sample is overloaded Be sure to load the recommended volume of sample from the wells per well Use Use the Two Step Loading method page 126 Two Step Use the Two Step Loading method page 126 method page 126 Well
161. y Gently remove the comb from the gel Load the samples in the wells of the E Gel as described on page 32 Proceed to running the gel below 1 Run the gel at 60 70 volts constant voltage or 40 50 mA constant current for 30 minutes for single comb gels or 15 minutes for double comb gels Do not run longer than 45 minutes single comb gels or 25 minutes double comb gels Longer run times will damage the gel Do not allow the current to exceed 60 mA Turn down the voltage to decrease current 2 Atthe end of the run remove the gel cassette from the power unit and analyze your results on a UV transilluminator Downloading Firmware Upgrades for the E Gel iBase Introduction iBase Updater Firmware Update Troubleshooting Instructions are provided below to upgrade the firmware on the E Gel iBase Power System Firmware upgrade requires installation of the iBase Updater program 1 Download the iBase Updater file iBaseUpdater zip from www invitrogen com ibase Extract the iBaseUpdater exe file from the zip folder Double click the iBaseUpdater exe file and follow the instructions to install the program To launch the iBase Updater click on Start gt All Programs gt Invitrogen gt Updaters and select iBase Updater Pew Ne Disconnect the electrical plug of the iBase from the electrical outlet Make sure the USB cable is not connected Press and hold the Go button red button Conti
162. y When bound to nucleic acids the proprietary nucleic acid stain in E Gel EX and E Gel SizeSelect agarose gels has fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm see figure below Normalized fluorescence excitation and emission spectra of proprietary DNA gel stain in E Gel EX and E Gel SizeSelect agarose gels determined in the presence of DNA Excitation Emission Fluorescent excitation Fluorescence emission 350 450 550 650 Wavelength nm Detect DNA bands stained with proprietary DNA gel stain using a blue light transilluminator a standard UV transilluminator or a laser based scanner For photographing gels a special filter may be required refer to page 51 for more information Using a blue light transilluminator method dramatically reduces DNA damage As a result cloning efficiency can improve ten to thousand fold For more information see the brochure http www invitrogen com content sfs brochures 713 01825 011203 EGel bro pdf 21 Safe Imager Blue Light Transilluminators Advantages of Blue light Transillumination Instrument Specifications AN Caution Introduction 22 Unlike UV transilluminators the Safe Imager transilluminators do not produce UV light which results in the following advantages e The Safe Imager does not require UV protective equipment during use e Blue light transillumination results in dramatically increased
163. y Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 1 minute of sample immediately after sample loading loading Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current time or high current during resulting in poor band migration or a melted gel Do the run not run the gel longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 15 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well the recommended Load the recommended sample volume per well volume per well from the wells Use the Two Step Loading method page 126 Wells damaged during comb Remove the comb gently without damaging the wells removal Continued on next page 68 Troubleshooting Continued Problem RNA sample cannot be seen Failure Mode indicated by continuous rapid beeping and Cassette Missing Hold Go to Reset or a steady red light Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency Cause Inhibition of visualization by

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