Rotaphor VI user manual
Contents
1. 6 8 3 1 Content eg 8 3 1 1 Electrophoresis chamber 8 72 Gelcasting VANS ERR t m 8 lsd ee ul e 8 3 1 4 Powersupply rtis xe t dria d oci ee re a REESE Re ad 8 3 1 5 Block el 8 3 2 Unpack and check 8 3 3 Installation conditions ssssssssssseem nennen nennen 9 3 4 Setup of the thermostatic 9 3 5 Setup of the computer esee nenne there nnn rear n nennen nenne 9 3 6 Setup of the power 9 3 7 lee C tm 10 Experimental Considerations leeeeeee esee eeeeee eee ee eene nnmnnn nnmnnn nna 11 4 1 11 4 2 ele LE 11 MN Eer E E 11 422 e EE 12 4 2 3 Parameter editor 13 4 2 4 Main screen during the 22 0 15 4 3 Pre set standard parameter leie eene 17 4 4 Conventional gel electrophoresis with the Rotaphor System NEN NE 17 4 5 Consequences of parameter modifications 17 4 5 1 Angles between 91 and TOU EE 19 Materials and methligds iiuuiusis iussa nic i i nn SE ERREUR KD xanax CH ER E Ea x RUOLO ROT E 21 5 11 82. 35 11 Note for the disposal of electric electronic waste eee 37 12 Warka MY emt 38 WE EE 39 Trouble Shooting 40 14 1 Rotaphor does not function 40 14 2 Experimental problems nnne 41 Biometra An Analytik Jena Company 1 Introduction 1 1 Field of applications The Rotaphor System is meant to be used for the separation of very large DNA molecules in agarose gels by pulsed field electrophoresis PFGE 1 2 Special features 1 2 1 Rotating Field Electrophoresis ROFE In contrast to conventional PFGE instruments with fixed electrodes the patented Rotaphor system features free rotating electrodes Thus any angle can be set for the electric field Changing field angles over the time are achieved by the precise movement of the rotor This feature is of special importance for the separation of very large DNA molecules 1 2 2 Easy to program easy to run The Rotaphor Version 6 0 provides a convenient computer software for programming new experiments and electrophoresis control Seventeen pre set programs are available for various separation ranges and are an ideal starting point for new experiments For each protocol a real gel picture is shown in the software individual pictures for custom protocols can be uploaded 1 2 3 Integrated buffer circulation The Rotaphor system
3. 31 10 2012 16 53 08 Rotor moves to default position 31 10 2012 16 53 08 Lid closed Edit Start Electrophoresis Parameter Lists Start up Tests running No list was selected iz Figure 3 Rotaphor Software main screen 4 2 3 Parameter editor To access the parameter editor press button Edit Parameter Lists in the main window see Figure 6 In the parameter editor 9 preset lists for different sizes of fragments are available 1kb 100kb 14h 2kb 200kb 18h 3kb 250kb 21h 1kb 450kb 23h 2kb 800kb 24h 3kb 1600kb 24h 20kb 2600kb 48h Okb 5700kb 120h Okb 5700kb 240h CO NI o0 Nj Each preset parameter list has been tested thoroughly and for each list a gel picture is displayed that gives an impression about the results that can be expected In the Comments field useful additional information on the used buffer type and concentration and the agarose concentration is given Please note that the preset parameter lists are marked by a sign and cannot be deleted Note For those customers who use the instrument for the first time we strongly recommend to start with a preset parameter list before programming individual parameter lists Biometra An Analytik Jena Company Parameter E ditor Help F1 User markus Parameter Lists Lists of other users backups mck 1 Name of List 2kb 800kb 24h user selected 1kb 100kb 14h 2kb 200kb 18h S EEN D
4. 21 5 2 Buffers and solution 21 5 2 1 Stock solution of TE 21 5 2 2 Stock solution of Loenning running 21 den 22 5 24 PI ug 22 52 5 TE DUffer i essem tr Re ERR RR ERE RIPE KC ERR EUR RO EH HER LER Ra YR RR VR Ra 22 5 2 6 TAE buffer 1X EE 22 5227 WBE buffer O 5X EE 22 Instruction Manual Rotaphor 5 2 8 EC lysis TEE ER 529 ene Ee EE ER 5 2 10 PMSF Selten ee Ee EE 23 6 Sample ee ere teehee ee dE 24 6 1 Preparation Of agarose plugs ren E ru 24 6 1 1 5 cerevisiae sample preparation 24 6 1 2 coli sample 25 6 1 3 Mammalian cells sample preparation 26 6 1 4 Restriction enzyme treatment of agarose 5 26 EAS UC Dope EET ER 28 7 1 EET 28 7 2 Setting up the electrophoresis chamber 28 7 3 Freet Eer Ee ens E Den Fev e 28 E SEA cic e 28 7 32 B lffer concentratiori EE 28 7 33 General 28 7 4 Preparation of agarose 9816 teet ette 29 8 EN 33 EE 34 9 1 Instructions for return 34 10 Equipment Decontamination
5. Biometra distributor Biometra GmbH Service Department Rudolf Wissell Straf e 14 16 D 37079 Gottingen Phone 49 0 5 51 50 68 6 10 or 12 Fax 49 0 5 51 50686 11 e mail Service biometra com 9 1 Instructions for return shipment In case of an instrument failure that cannot be fixed by the procedures described in section Fehler Verweisquelle konnte nicht gefunden werden please proceed as follows e Return only defective devices For technical problems which are not definitively recognisable as device faults please contact the Technical Service Department at Biometra Tel 49 551 50881 10 12 Fax 49 551 50881 11 e mail service biometra com Please contact our service department for providing a return authorization number RAN This number has to be applied clearly visible to the outer box Returns without the RAN will be not be accepted Important Carefully clean all parts of the instrument of biologically dangerous chemical or radioactive contaminants If an instrument is contaminated Biometra will be forced to refuse to accept the device The sender of the repair order will be held liable for possible losses resulting from insufficient decontamination of the device Please prepare written confirmation that the device is free from biologically dangerous and radioactive contaminants The declaration of decontamination see section 10 must be attached to the outside of the packaging Use the original p
6. Proceed as usual with staining destaining nicking blotting and hybridization Note that special care has to be taken to prepare the gel for blotting when very long DNA molecules have been separated We have found that vacuum blotting gives an excellent sharpness after transfer but the sensitivity of hybridization is better with capillary blotting After each electrophoresis experiment the chamber should be emptied in the following way Insert one half of a tubing connector of the appropriate diameter into a long piece of silicone tubing and press the other half into the outlet of the pump The free end of the tubing should hang into a large container standing on the floor Switch off the pump at the controller when the tube is filled with buffer Remove the buffer by gravity siphoning into the container Moving around the upper end of the tubing helps cleaning the chamber and the inlet of the pump from agarose particulate matter At the end of the draining procedure the instrument should be tilted to remove the last small volumes of buffer In order to prepare the tray for the next gel remove the agarose plugs from the 4 cylindrical holes with a spatula or by vigorously blowing air into the holes Check that the inlet of the buffer circulation pump is free of all particulate matter If the instrument is not to be used immediately let it air dry with the lid removed from the main chamber body If the instrument is not to be used for more than 1
7. logarithmic to see Figure 5 for the differences For the angle 0 additionally the mode PFGE exists 14 Instruction Manual Rotaphor ei Constant Linear to Logarithmic to Figure 5 Modes available to define the course between two parameter values If necessary enter comments in the field Comments To save the parameter list press Accept changes To delete a parameter list press Delete Parameter List Tipp For the separation of small fragments 25 kb or smaller the checkbox beside Duration has to be activated Duration h 14 With activated checkbox current is applied also during rotor movements This ensures a good separation of very small fragments To load a parameter list of another user use the drop down menu Lists of other users backups Select the user from the drop down menu and then select one of the loaded parameter lists For each custom parameter list a gel image can be assigned Press Assign Image and select a jpg or gif file to be uploaded gel image is linked to the currently activated parameter list In this way a database of parameter lists and corresponding gel images can be created The Rotaphor Software also allows to combine parameter lists Combined parameter lists are useful whenever parameters should undergo several changes during the run The single lists are run consecutively by the instrument In the parameter editor 8 preset combi
8. 12 00 18 08 2004 12 00 47 Electrophoresis started 18 08 2004 12 00 45 Rotor in default position Edit 18 08 2004 11 58 18 Rotor moves to default position Interrupt 18 08 2004 11 56 18 Lid closed Parameter Electrophoresis Lists Start up Tests running 2kb 600kb24h P Figure 6 Main screen during run The main screen provides three checkboxes for user interaction Control Temperature control on or off The temperature temperature set in the parameter list is shown in the field Set Temperature C and the measured buffer temperature in big digits above The Rotaphor System automatically controls the buffer temperature in the electrophoresis chamber by a valve The control function for the buffer temperature can be switched off Pump Internal buffer pump on or off For the application of liquid samples the pump has to be switched off The pump can be switched on manually 10 15 min after sample application or switches on automatically after 30 min Alarm Alarm on or off The alarm function gives an acoustic signal in case of a failure A running electrophoresis can be paused at any time Press Interrupt Electrophoresis to pause the run and Continue electrophoresis to continue a paused run 16 Instruction Manual Rotaphor 4 3 Pre set standard parameter lists The controller software includes numerous parameter lists covering most of the separation needs
9. After selection of such a parameter list an image of a separation of different size markers will be shown that was generated using the respective parameters These images are intended to provide a realistic idea of the inherent separation performances and should act as guidelines for initial separation conditions for a given DNA size range If you need to fine tune the separation conditions we recommend that you start with a standard list and apply subtle changes Please note that an alteration of only one parameter mentioned above will lead to a different behaviour of DNA molecules in the gel with obvious consequences for their separation If for instance the voltage is increased by 2 Volts at the separation of S pombe the upper bands fade or become invisible see below 4 4 Conventional gel electrophoresis with the Rotaphor System It is also possible to run a conventional electrophoresis with the Rotaphor In this case place the gel support tray with gel and corner insulators into the buffer filled electrophoresis chamber and wait until buffer has reached the predetermined temperature Stop buffer circulation by turning of the pump by unchecking the box Pump in the main window Otherwise the buffer circulation will wash your samples out of the wells Enter the DNA in sample buffer e g 1096 glycerine directly into the wells of the gel Close the lid very carefully choose for start and end angle 0 if you want to apply this electrophor
10. SCH vectors is more obtuse at PFGE 2 55329 Consequently the force backwards O direction is larger that in forwards direction Therefore the trailing end becomes the leading end and the whole molecule if forced through the pore which harboured this end when the field was last changed Since the trailing end Pp 3 Q of a short molecule is not as far behind as the trailing end of a larger DNA molecule the shorter molecule has lt moved further than larger molecule The position difference A results in the observed separation of the respective molecules This simple theory allows the prediction of the consequences of parameter changes which fit the observed O behaviour very closely at least if only subtle changes are applied 1 Duration The duration of an electrophoresis is directly proportional to the distance a given DNA molecule moves during a run if no exponential voltage ramps are used The separation distance A increases proportional to the travelling distance and therefore also proportional to the duration In reality it has to be taken into account that a gel has a limited size and bands which have left the gel are lost 2 Interval According to the theory shown above the separation distance A increases with the number of pulses Since separation takes place only if the lagging ends of all molecules that should be separated have left the pore of the pulse before th
11. U of restriction enzyme 20 ul of appropriate 10x restriction buffer BSA if required add acetylated BSA at a concentration recommended for the particular restriction enzyme as many BSA preparations are contaminated with nucleases check using restriction buffer and test 0 02 Triton X 100 agarose chops and sterile water to make up the volume Instruction Manual Rotaphor Biometra An Analytik Jena Company Gently shake the plug at the recommended temperature overnight CRITICAL STEP Proper controls must be performed all components of the digestion mixture including the chops themselves must be checked for the presence of endogenous nuclease activity This can be achieved by setting up mock digestions with chopped plugs and restriction buffer Each additional reaction component can be added to a separate sample and incubated at proper temperature for a few hours Running PFGE on these samples will reveal nuclease activity as smeared bands or a smear of high molecular weight DNA entering the gel matrix Commercially purchased acetylated BSA occasionally contains nuclease 8 To stop the reactions remove the digestion mixture and replace with 1 ml NDSK Shake on ice for 2 h Store reaction products indefinitely in either NDSK buffer or 0 5 M EDTA pH 9 5 at 4 C or proceed with electrophoresis CRITICAL STEP Digestions in agarose are typically robust Problems are usually traced to inhibitors and contaminants indicative of a
12. cells by centrifuging at 1 000g for 15 min at 4 C b Discard the supernatant and wash the cells twice in 0 05 M EDTA pH 7 5 spin down the cells at 1 000g for 15 min at 4 after each wash keeping the samples on ice during washes 24 Instruction Manual Rotaphor d f g h i 6 1 2 b c d e f g h Biometra An Analytik Jena Company Count the cells using any reliable method CRITICAL STEP When resuspending pellets after the final wash and or count keep in mind that this solution will be diluted 1 1 into agarose Calculate cell concentration and resuspension volume accordingly Heat a 1 2 solution of LMP agarose in 0 125 M EDTA pH 7 5 and equilibrate to 42 50 C in a waterbath This solution can be held until ready for use Mix the cells and agarose together Mix the cells with an equal amount of agarose Also add 15 5 ul of Zymolyase solution per ml of agarose cell suspension CRITICAL STEP Warm the cell suspension briefly in a 37 C waterbath before mixing with agarose When mixing take care to pipette thoroughly but slowly to avoid damaging the cells and shearing the DNA Keep the agarose cell suspension in a beaker of warm water on the benchtop to avoid premature gelation Pipette the agarose cell suspension into a casting mould taking care to avoid air bubbles Allow the moulds to set at 4 C until the agarose has gelled After solidification of the low me
13. of the wells in the final agarose gel When the agarose blocks are prepared with the sample mould provided they will match the size of teeth of the comb delivered with the Rotaphor System Before filling up the holes of the mould moisten the mould by immersing it into water this avoids air bubbles at the corners Dry the surface and seal one side of the mould with water resistant adhesive tape Alternately agarose plugs can be prepared by simply filling a small 1ml syringe with the tip cut off or trough with the agarose cell mixture and cutting the resulting brick into slices that approximate the volume of the wells 2 Obtain sample material from any desired source including bacterial cultures yeast cultures mammalian cultured cells lymphocytes frozen tissue etc A good starting point for genomic DNA concentration is 1 2 ug per plug but this value can vary substantially depending on the application of a particular PFGE experiment Approximate cell counts for 2 ug genomic DNA plugs are 6 x 10 cells per plug for S cerevisiae DNA genome size 12 Mb 2 x 10 cells per plug for coli DNA genome size 5 Mb and 3 x 10 cells per plug for mammalian DNA genome size 3 000 Mb 3 Separate the cells from storage or growth media and wash to remove debris and prematurely lysed cells Follow the following instructions for sample preparation for S cerevisiae E coli or mammalian cells 6 1 1 5 cerevisiae sample preparation a Pellet
14. or other parts are visible Unplug the power cable before you open the Rotaphor System Danger of electric shock Make sure that the appliance connector and the plug of the supply cord are accessible so you can separate the instrument from the mains Connect the Rotaphor System to a grounded socket Appropriate safety regulations must be observed when working with infectious pathogenic or radioactive material Ask the responsible local safety inspector for details The Rotaphor System must not be used with explosive flammable or volatile liquids Instruction Manual Rotaphor This instrument is designed and certified to meet EN 61010 1 safety standards It should not be modified or altered in any way Alteration of this instrument will void the warranty void the EN61010 1 certification and create a potential safety hazard Place the Rotaphor System on a stable non flammable surface in a dry safe environment Do not use alcohol e g methanol ethanol organic solvents or abrasives to clean the instrument For transports always use the original Biometra box Biometra An Analytik Jena Company 3 Installation 3 1 Content of delivery 3 1 1 3 1 2 3 1 3 Electrophoresis chamber Lower part with data and power cable Safety lid with Rotor V2 1 Gel support tray 4 corner insulators Note Each Rotaphor System is checked individually and therefore faint scratches on the gel support tray may occ
15. week pipette 0 5 ml glycerine into the pump inlet for pump maintenance 32 Instruction Manual Rotaphor An Analytik Jena Company 8 Maintenance The Rotaphor electrophoresis chamber is designed to operate with aqueous solutions and not with organic solvents Do not run the buffer circulation pump without buffer in the electrophoresis unit since the pump is not designed to run dry The pump motor with the impeller is fitted to the pump hose through a bayonet attachment and can be changed if necessary It is not essential to clean the electrodes or any other part of the lid routinely but it is essential to empty the chamber after each run Do not close the Rotaphor until chamber tray and lid are dry Very high humidity e g generated by buffers in the closed chamber at room temperature leads to condensation on the metallic parts of the electronics and can therefore lead to early age hardening If the Rotaphor electrophoresis chamber is not to be used for a long period of time it should be rinsed several times with tap water and finally once with deionised water To dry leave the electrophoresis chamber upside down without the lid for a suitable period of time and then pipet 0 5ml glycerine into the pump inlet before storing it away Instruction Manual Rotaphor 33 Biometra An Analytik Jena Company 9 Service Should you have any problems with this unit please contact our service department or your local
16. Incorrect buffer concentration used Use correct buffer Incorrect buffer pH used Use correct buffer Lanes are distorted The electrophoresis chamber is not exactly horizontal or the lid is incorrectly positioned on the chamber Check level with large spirit level without the lid in place on top of distance pillars Check current from power supply The current should be the same in both rotor positions Corner insulators have not been in place during electrophoresis or have been mounted incorrectly Incorrect volume of buffer used Check that volume is 2 4 liters Poor grade agarose employed in experiments Poor circulation of electrophoresis buffer The pump does not work as described above o The fuse inside the computer is blown Replace with 800mA mtr fuse o Theinlet of the pump may be blocked with debris o Check connector at the rear of the computer Instruction Manual Rotaphor 41 Biometra An Analytik Jena Company 42 o If you did not apply glycerine before long time storage crystals have formed in pump Try to solve these blocking crystals with water over some days or contact your dealer o Inlet of the pump is blocked Clean it and take care when preparing the gel that all loose agarose particles are removed before the gel tray is placed in the chamber Air bubbles sucked under the tray by very intensive buffer circulation do not affect the separation Different velocity of same DNA si
17. P agarose for preparative gels 5 2 Buffers and solution Please note that best electrophoresis results are usually obtained with 0 025 M TBE pH 8 5 running buffer 5 2 1 Stock solution of TBE running buffer To prepare 1 stock solution usually known as 10x TBE dissolve the following substances in 900 ml deionized or distilled water e 108g Tris 55 g Boric Acid 40ml 0 5 M EDTA pH 9 Adjust volume to 1000 ml IMPORTANT Store this stock solution at 4 C and do not keep it for more than two weeks The electrophoresis buffer is obtained by diluting the stock solution 1 40 with distilled water 0 025 x TBE 60 ml 2400 ml Pour 2 4 of this buffer into the electrophoresis chamber 5 2 2 Stock solution of Loenning running buffer To prepare 1 stock solution dissolve the following substances in 900 ml deionised or distilled water 44g Tris e 42g NaH PO e 20ml0 5 M EDTA pH 7 5 Adjust volume to 1000 ml Store this stock solution at room temperature Instruction Manual Rotaphor 21 An Analytik Jena Company The final electrophoresis buffer is obtained by diluting the stock solution 1 30 with distilled water 80 ml 2400 ml Pour 2 4 of this buffer into the electrophoresis chamber IMPORTANT This buffer withstands only an electrophoresis duration of less than 30 hours but small DNA fragments 20 100kb resolve very well The current with this buffer is very high Therefor
18. Rotaphor System 6 0 Instruction Manual Model Ord No Rotaphor System 6 0 230V 021 100 Rotaphor System 6 0 110V 021 190 Rotaphor System 6 0 230V 021 200 Please read these instructions carefully before using this apparatus Biometra An Analytik Jena Company Rudolf Wissell Str 30 Service Department D 37079 G ttingen Rudolf Wissell Strasse 14 16 Tel 49 551 50686 0 D 37079 G ttingen Fax 49 551 50686 66 Tel 49 551 50881 10 12 email info biometra com Fax 49 551 50881 11 Internet http www biometra com email service biometra com Biometra An Analytik Jena Company 1 2 3 4 5 M 4 1 1 Field of applicatiorisii t n cantactande PR XR ERES 4 1 2 Special TOALUES ce 4 1 2 1 Rotating Field Electrophoresis 4 1 2 2 Easy to program easy ausube nut utu dud er oup tns 4 1 2 3 Integrated buffer circulation E 4 1 3 Technical 4 1 3 1 Electrophoresis 4 iO EN E A 1 9 3 Control UNT EE 5 1 3 4 Rotaphor 6 0 5 1 3 9 KEE DEE 5 GECKEN 6 2 1 Definition et E 6 2 2 General Safety
19. acking material If not available contact Biometra or your local distributor Label the outside of the box with CAUTION SENSITIVE ELECTRONIC INSTRUMENT Please enclose a note which contains the following a Senders name and address b Name of a contact person for further inquiries with telephone number c Description of the fault which also reveals during which procedures the fault occurred if possible 34 Instruction Manual Rotaphor An Analytik Jena Company 10 Equipment Decontamination Certificate To enable us to comply with German law i e 871 StriSchV 817 GefStoffV and 819 ChemG and to avoid exposure to hazardous materials during handling or repair please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS PHONE NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Method of cleaning decontamination The equipment has been cleaned and decontaminated NAME POSITION HEAD OF DEP INSTITUTE COMPANY SIGNED DATE Instruction Manual Rotaphor 35 Biometra An Analytik Jena Company PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER General Informatio
20. ams can be linked to create complex separation pattern Program in or decremental field vectors linear or logarithmic 0 or 95 160 Program in or decremental pulse length 1 10 000 s Program voltage for electrophoresis 30 225 Volt 1 8 5 V cm Precise control of buffer temperature 5 22 C For each program a real gel picture can be loaded displayed Convenient user administration Comprehensive online help German or English language Printed user manual Power Supply Rotaphor 6 0 interface Voltage 0 300 V Current 0 450 mA Dimensions 5 2 cm x 22 2 cm x 20 2 cm H x W x D Weight 1 7 kg Biometra An Analytik Jena Company 2 Safety and Warning Notices 2 1 Definition of Symbols Symbol PPP Definition Caution Refer to instruction manual Danger High voltage Fragile 2 2 General Safety Instructions Please read this manual carefully before starting operation of the Rotaphor System General safety precautions for laboratory work must be observed when working with the Rotaphor System The Rotaphor System does not produce a sound power level that could be hazardous for the user A The Rotaphor System contains no user serviceable parts Do open the instruments housing Service and repair may only be carried out by the Biometra Service department or otherwise qualified technical personal Do not use the instrument when damages of the housing cable
21. anguage Please choose a directory for your data or accept the default o Do not create user account Figure 2 Create a user account To cancel the creation of a new user account press Do not create user account 4 2 2 Main screen The Rotaphor Software main screen displays all important parameters during the run see chapter 4 2 4 Additionally the main screen features a menu to administrate the Rotaphor System Options User Administration Can be used to create new user accounts or to change individual settings like password and language All changes have to be confirmed by Save Changes Temperature Calibration Before delivery the Rotaphor System is calibrated thoroughly However if the temperature sensor should be misaligned a recalibration is possible For this purpose the buffer temperature in the electrophoresis chamber has to be measured at room temperature and the measured value to be entered in the field Rotor Speed If necessary the rotor speed can be adjusted between 0 and 9990 Logbook The logbook records all actions and messages during a run It is a useful tool for failure analysis Exit Closes the Rotaphor Software Help Help Comprehensive help function for the Rotaphor Software About Provides information on the software version and serial number 12 Instruction Manual Rotaphor Temperature 1 5 Control Temperature v Pump Alarm 2
22. as remover provided with the apparatus For convenience cut the blocks with a scalpel into two identical parts and transfer them into a container filled with NDS buffer If the agarose cell suspension was poured in a syringe as a brick slice the brick into appropriate pieces Incubate the plugs in NDSK for two nights at 50 C to create spheroplasts disrupt membranes and digest other cellular debris Ideally use a 50 ml conical tube and at least 2x the volume of the plugs of buffer CRITICAL STEP In general more buffer is better but some reagents can be costly Store agarose plugs from any cell type in either NDSK buffer or 0 5 M EDTA pH 9 5 If kept at 4 C the plugs and chromosomal DNA will remain intact for years without any appreciable degradation 6 1 4 Restriction enzyme treatment of agarose plugs Please note that the enzyme treatment will take 3 days 26 4 5 Place the plugs in containing 0 01 mM PMSF use several milliliters of solution plug Incubate overnight at room temperature 20 25 C with gentle shaking Dialyze the plugs into a larger volume of TE place plugs into a sterile 50 ml conical tube filled with chilled buffer Gently shake or rock the plugs at 4 and make several buffer changes over the course of at least 24 h Prepare agarose chops by mincing a plug into smaller 0 5 1 mm pieces using a sterile glass coverslip Prepare digestion mixture total volume 200 ul by combining 50
23. ctor of the cable to the chamber is inserted correctly at the rear of the computer The buffer circulation pump does not work Instruction Manual Rotaphor An Analytik Jena Company The checkbox Pump in the main window is not checked Click the checkbox to start buffer pump Relay becomes faulty with age Contact your dealer for replacement Check whether the connector of the cable to the chamber is inserted correctly at the rear of the computer Platinum electrodes do not have shiny metallic finish after electrophoresis The water is insufficiently deionized Use distiled or double distilled water to prepare buffer Chemicals are not of the required quality Use only analytical grade chemicals Build up of condensation on the lid during or after the run Buffer temperature is too high Selected temperature limit is too high The buffer temperature should be always 5 C lower than the air temperature around the Rotaphor System 3 way valve is malfunctioning you hear no clack from the chamber when the valve is switched on off Thermostatic circulator does not work or chosen temperature limit is too high After completion of electrophoresis unit was not sufficiently dried If there is any problem not mentioned here please contact your dealer 14 2 Experimental problems a b Very large DNAs do not enter the gel Field strength is too high Use lower field strength see explanation above
24. e do not apply voltages above 130V otherwise the rotor electronics might be damaged or the fuse will blow 5 23 TNE buffer 10 mM Tris e 200 mM NaCl e 100 mM EDTA pH 7 2 5 2 4 NDSK buffer 0 5 e 1 w v N laurylsarcosine e mg ml 1 Proteinase K pH 9 5 Solution may need to be heated to 50 to dissolve components 5 2 5 TE buffer 10mM Tris e 1 mM EDTA pH 8 0 5 2 6 TAE buffer 1x 40mM Tris acetate e 1 mM EDTA pH 8 0 5 2 7 TBE buffer 0 5x e 44 5 mM Tris borate e 1mM EDTA pH 8 3 5 2 8 1 515 buffer 6mM Tris HCl pH 7 6 e 1M NaCI w v e 100 mM EDTA e 0 5 w v Brij 58 polyoxyethylene 20 cetyl ester 0 2 w v deoxycholate 0 596 w v N laurylsarcosine e 1 mg ml 1 Lysozyme 2019 ml 1 RNase 5 2 9 Zymolase solution Add Zymolyase 20 T to glycerol phosphate buffer 10 mM sodium phosphate and 50 v v glycerol 7 5 at a concentration of 1 mg ml 1 22 Instruction Manual Rotaphor An Analytik Jena Company Store at 20 C 5 2 10 PMSF solution e Prepare a 0 1 mM solution of PMSF phenylmethanesulfonyl fluoride in isopropyl alcohol Store at 20 C Instruction Manual Rotaphor 23 Biometra An Analytik Jena Company 6 Sample Preparation This protocol is designed to provide a basic introduction to sample preparation and techniques that are unique to PFGE Sample preparation for PFGE is fundamentally different from s
25. e gel becoming detached from the gel tray during electrophoresis Avoid an uneven gel surface see below Remove all loose agarose particles from the tray and close the holes of the 4 screws in the tray at the lower side with water resistant adhesive tape to avoid any possible distortion of the electric field due to electricity flow through these holes Note Pulsed field gel electrophoresis is not compatible with ethidium bromide in the gel during the run DNA becomes stiff and moves very badly if the dye is intercalated Consequently the separation range is narrowed dramatically Instruction Manual Rotaphor 29 An Analytik Jena Company Comb Adhesion pulls up the agarose solution at the casting frame and at the comb This causes an uneven gel surface i S T O 8 Gel support tray Agarose filled slot Especially after filling and Buffer capping the slots the buffer circulation above the gel is blocked by this agarose rampart DNA sample Buffer circulation gt d dee Therefore cut away agarose rampart with scalpel to allow free buffer circulation 3 6 Loading the gel To load the gel cut the agarose plugs containing the sample to the desired size and insert them into the wells using a metal spatula Make sure that the plugs attach to the front wall direction of electrophoretic separation of the well and finally seal the wells wi
26. e product of interval time and field strength voltage needs to be adequately high Otherwise the larger molecules are trapped in a pore do almost not move and are not separated It might therefore be concluded that increasing interval and or voltage increases the upper border of the separation range Since in both cases the number of pulses per run is reduced higher voltages result in shorter duration the separation distance between two given molecules is also reduced Biometra An Analytik Jena Company 3 Angle According to the above theory the angle should be not relevant for the separation quality if it is larger than 90 Only the duration needed for the same travelling distance should increase with the angle This fits more or less the observed situation but more obtuse angles result in sharper bands It therefore needs to be decided whether short runs or sharp bands are most relevant 4 Voltage Most of the influence of the field strength has already been discussed together with the interval but there is an observation which needs to be mentioned If the DNA molecules are larger than 2000 kb a phenomenon called trapping leads to impaired results Trapping means that a voltage dependent fraction of the molecules is trapped each pulse in the gel If huge amounts of such DNA are applied to the gel these trapped molecules may be seen after staining as smeary ladder Consequently the amount of DNA in the real band is red
27. ecommend it since only the electrode power supply from Biometra has a suitable interface This interface enables the computer to control the field strength automatically and to switch of electrode voltage if no electrophoresis is running Biometra An Analytik Jena Company 3 7 Final Settings Use the levelling feet to maintain a horizontal position of the electrophoresis chamber Because it is very important for optimal results measure the horizontal position of the electrophoresis chamber at the top of the safety lid at the 3 possible sides with a large spirit level and correct the position if necessary with the levelling feet Connect the power cords to the rear of the computer the monitor and the power supply Finally connect the power leads to a properly grounded wall socket 10 Instruction Manual Rotaphor 4 Experimental Considerations 4 1 Introduction The search for a theoretical basis regarding the separation of very large DNA by pulsed field gel electrophoresis revealed that no simple theory describes the all the characteristics of this method We will therefore use a relatively simple model to define parameters for separations of the desired size range In all cases the following parameters have to be considered e Size of the nucleic acid molecules e Interval e Angle between the orientations of the electric field e Field strength e Temperature e Agarose concentration lonic strength of the electrophoresis bu
28. esis as 274 dimension after a please enter 90 for start and end angle Please note that the DNA moves from right to left in this second dimension Enter also a suitable duration and field strength and start electrophoresis Since inhomogeneous temperatures impair the results the buffer circulation pump will start after 30 minutes automatically 4 5 Consequences of parameter modifications Using a simple theory developed by Southern and colleagues the consequences of modifications made to parameter lists shall be discussed This theory explains relatively well the observed changes of DNA mobility if applied only qualitatively and if parameters are not changed dramatically Biometra An Analytik Jena Company re 18 wi i O SS Two DNA molecules are shown a large DNA 300kb and a small one 50kb These move driven by the electrical filed through the gel pores After changing the field angle by 90 or less the DNA molecules will move into the new direction After direction change the leading end is still the leading end Since velocity of DNA molecules with a size above 50 kb is virtually identical no separation of the small and large molecules will be observed Instruction Manual Rotaphor 4 5 1 Angles between 91 and 160 Looking at the pulsed gel electrophoresis the situation is identical e at the beginning gt However the angle between the field
29. features an internal pump for efficient buffer circulation Buffer flow from an external refrigerated circulator and internal buffer flow are completely separated An integrated thermostat relay precisely monitors the buffer temperature and triggers if necessary the cooling circuit 1 3 Technical specifications 1 3 1 Electrophoresis chamber Freely rotating electrode rotor with 14 platinum electrodes 160 max angle Integrated buffer circulation pump Internal cooling circuit for connection of an external thermostat Temperature sensor 2 4 buffer volume Acrylic glass safety lid automatic shut off when lid is lifted Dimensions 35 cm x 47cm x 25cm W x D x H 1 3 2 Geltray Adjustable feet for horizontal levelling 20 x 20 cm gel format 18 cm separation distance 18 teeth comb 2 0 mm optional 40 teeth Gel casting frame Moulding form for 20 agarose embedded samples 4 Instruction Manual Rotaphor 1 3 3 b e e e e e e e P Gei Control unit Up to date Personal Computer with Windows operating system Control interface for Rotaphor electrophoresis chamber Installed Rotaphor 6 0 control software Rotaphor 6 0 Software 17 optimized programs for separation of samples from 100 kb up to 6 Mb Up to 11 programs can be combined and run subsequently 8 pre installed combined program lists Easy creation of new programs angle Voltage Pulse length Maximum program length 500 hours Progr
30. ffer e Duration of electrophoresis 4 2 Software The Rotaphor System is controlled by the Rotaphor Software The software features a user management and demands the user to login after program start After login the software is started that is divided into two main screens In the parameter editor parameter lists are programmed and in the main window the applied parameters and messages are shown 4 2 1 Login Screen After starting the Rotaphor Software at first a user account for login has to be selected see Figure 1 Select a user from the displayed list enter the user specific password and press Select user account User Selection 4 3 Please select user and enter respective password Anwender Zwei markus markus deutsch Selected language Password Select user account Create user account Figure 1 Login Screen An Analytik Jena Company To create a new user account press Create user account In the next screen see Figure 2 enter a user name select a language and define a password optional Personal data will be saved in the preset directory Optionally an alternative path and directory can be set 4 Create a new user account Please enter username password and a directory name The respective directory will hold your data files You may also accept the default directory The declaration of a password is optional Default User Ji Password Select l
31. gher than 255 V IMPORTANT Never apply voltages higher than 255 V to the Rotaphor System 7 3 2 Buffer concentration The Rotaphor System is designed to work with the buffer concentrations described above Working with buffer concentrations higher than this leads to a high current Consequently the electronics generates a considerable amount of heat especially at high voltages which may not be carried off properly At 80 C a fuse inside the rotor or the fuse at the back of the controller will disconnect the rotor from the electricity IMPORTANT Never apply buffer concentrations that lead to a power consumption above 40 W 7 3 8 General precautions Some ions e g chloride will lead to corrosion or other structural changes of the platinum electrodes These ions are a normal component of tap water IMPORTANT Use only deionized or double distilled water and chemicals of analytical grade Check quality of water and chemicals when electrodes do not shine metallically after a run The electronics of the Rotaphor System is only water resistant and not waterproof Particular care should be taken to minimize the periods of time the electronics are subjected to conditions of high humidity e g insufficient cooling of buffer during electrophoresis or storing the 28 Instruction Manual Rotaphor Biometra An Analytik Jena Company electrophoresis chamber filled with buffer and lid closed as this may over a considerable period of ti
32. ix the cells and agarose together Mix the cells with an equal amount of agarose CRITICAL STEP Warm the cell suspension briefly in a 37 C waterbath before mixing with agarose When mixing take care to pipette thoroughly but slowly to avoid damaging the cells and shearing the DNA Keep the agarose cell suspension in a beaker of warm water on the benchtop to avoid premature gelation Pipette the agarose cell suspension into a casting mould taking care to avoid air bubbles Allow the moulds to set at 4 C until the agarose has gelled After solidification of the low melting point agarose plugs 15 min remove the tape and push the blocks out of the mould by using the plexiglas remover provided with the apparatus For convenience cut the blocks with a scalpel into two identical parts and transfer them into a container filled with NDS buffer If the agarose cell suspension was poured in a syringe as a brick slice the brick into appropriate pieces Incubate the plugs in EC Lysis buffer overnight at 37 C to create spheroplasts disrupt membranes and digest other cellular debris Ideally use a 50 ml conical tube and at least 2x the volume of the plugs of buffer CRITICAL STEP In general more buffer is better but some reagents can be costly Instruction Manual Rotaphor 25 An Analytik Jena Company 6 1 3 i j a b d f g h Drain the buffer from the tube and replace with NDSK Inc
33. lting point agarose plugs 15 min remove the tape and push the blocks out of the mould by using the plexiglas remover provided with the apparatus For convenience cut the blocks with a scalpel into two identical parts and transfer them into a container filled with NDS buffer If the agarose cell suspension was poured in a syringe as a brick slice the brick into appropriate pieces Incubate the plugs 0 5 EDTA containing 7 5 5 mercaptoethanol overnight at 37 C to create spheroplasts disrupt membranes and digest other cellular debris Ideally use a 50 ml conical tube and at least 2x the volume of the plugs of buffer CRITICAL STEP In general more buffer is better but some reagents can be costly Drain the buffer from the tube and replace with NDSK Incubate overnight in NDSK at 50 C E coli sample preparation Pellet cells by centrifuging at 2 000g for 15 min at 4 C Discard the supernatant and wash the cells in TNE buffer twice spin down the cells at 2 000g for 15 min at 4 after each wash keeping the samples on ice during washes Quantitate the cells using any reliable method CRITICAL STEP When resuspending pellets after the final wash and or count keep in mind that this solution will be diluted 1 1 into agarose Calculate cell concentration and resuspension volume accordingly Heat a 1 2 solution of LMP agarose in dH20 and equilibrate to 42 50 C waterbath This solution can be held until ready for use M
34. me lead to corrosion of metallic components of the rotor electronics and chamber The Rotaphor System will be destroyed in that way IMPORTANT Please do not submerge the rotor electronics under water 7 4 Preparation of agarose gels Place the gel support tray on an even horizontal surface check with spirit level take away the corner insulators and secure the gel casting frame to the tray with the four screws provided To prepare a 196 gel of 7 mm thickness dissolve 3 0 g agarose in 300 ml of electrophoresis buffer Agarose gels from 0 5 to 1 5 can also be employed the choice of concentration depends the length of the nucleic acid molecules which are to be separated and the band sharpness desired Heat the suspension to boiling in a microwave oven ensuring that all agarose particles are properly dissolved Then for convenience seal the edges of the frame with agarose and let cool down the rest of the suspension to about 50 C before pouring the gel Then pour the gel and remove the remaining air bubbles from the 4 cylindrical holes and from the gel surface with a pipette At last position the comb It fits the two cylindrical holes on top of the gel casting frame Let the gel solidify for 45 min without moving it around When the gel is set carefully remove the comb and cut the gel from the casting frame with a scalpel Then remove the four screws and lift the frame very carefully Failures to take care at this point may result in th
35. n for Decontamination Please contact your responsible health amp safety officer for details Use of radioactive substances Please contact your responsible person for details Use of genetically change organism or parts of those Please contact your responsible person for details 36 Instruction Manual Rotaphor Biometra An Analytik Jena Company 11 Note for the disposal of electric electronic waste Note for disposal of electric electronic waste Hinweis f r die Entsorgung von Elektroaltger ten du traitement des d chets des appareils lectrique lectronique EE This symbol the crossed out wheelie bin means that this product should be brought to the Renseignement return and or separate systems available to end users according to yours country regulations when this product has reached the end of its lifetime For details please contact your local distributor This symbol applies only to the countries within the EEA EEA European Economics Area comprising all EU members plus Norway Iceland and Liechtenstein Dieses Symbol die durchgestrichene Abfalltonne bedeutet dass dieses Produkt von der Firma Biometra f r eine kostenlose Entsorgung zur ckgenommen wird Dies gilt nur f r Ger te die innerhalb Deutschlands gekauft worden sind Kontaktieren Sie f r die Entsorgung bitte die Biometra Service Abteilung AuBerhalb Deutschlands wenden Sie sich bitte an den lokalen H ndler Dieses Symb
36. nectors are correctly fitted Check that the lid has been correctly positioned Rotor does not stop at the light barrier In very brightly lit areas the light barrier can not work Operate only in normally lit areas and also avoid areas which may be subjected to direct sunlight The position indicator metal needle on the rotor is damaged Repair or replace needle The message Rotor Position inexact is often displayed In this case select a slower rotor speed The computer crashes regularly The computer may be subjected to considerable surges or electrical spikes This often occurs when numerous pieces of equipment are operating simultaneously in the same working area Change location of the instrument or filter the supply power There is no power at the electrodes The power supply is not working The fuse at the back of the chamber is blown Replace by a new 250mA 250V fuse Polarity is wrong Change the connectors at the power supply There is no contact between rotor electronics and the metal rings in the lid Withdraw rotor and bend metal pieces in a way that they make a good contact with the metal rings in the lid The 3 way valve of the temperature control does not work The TEMPERATURE LED does not light green main window Temperature chosen was too high Lower maximal temperature The TEMPERATURE LED lights green Relay becomes faulty with age Contact your dealer for replacement Check whether the conne
37. ned lists for different sizes of fragments are available As for the single parameter lists they are also marked by sign and cannot be deleted To start a run select a parameter list or combined parameter list and press Use Selected List for Experiment Them the parameter editor is closed and the main window is shown In the main window the name of the selected parameter list is displayed under the button Start Electrophoresis 4 2 4 Main screen during the run After starting a parameter list currently applied values for different parameters like interval angle and voltage are displayed The software additionally counts the remaining run time duration the time to the next rotor movement next move and provides information when the electrophoresis will be finished probably finished at The software records the status of all system components and messages regarding the electrophoresis by time and date in a message box In the message box also failures are displayed In addition with the logbook function the recorded messages are an important tool to monitor the electrophoresis and for failure analysis Biometra An Analytik Jena Company Sj Rotaphor V6 Demo Mode BBE Options Help Duration 23 59 35 Interval sec 60 9 Interval inverse sec 0 Angle 3 120 Set Temperature 1 3 Voltage V 180 Control Temperature vo Next move 60 Pump Probably finished at Thursday 19 8 2004 Alarm B
38. of methanol water or glycerine water coolant Connect the silicon tubing best with temperature isolation to the inlet and outlet of the electrophoresis chamber and secure the connections with hose clamps Connect the other ends of the inlet and outlet of the thermostatic circulator IMPORTANT Connect the outlet of the thermostatic circulator to the inlet of the electrophoresis chamber and the outlet of the electrophoresis chamber to the inlet of the thermostatic circulator 3 5 Setup of the computer Connect the following cables to the sockets at the back of the computer Keyboard connector encoded violet Mouse connector encoded light green e Monitor connector VGA connector it fits to the graphics card encoded blue e Cable of the electrophoresis chamber fits to the controller card installed at the computer e Data cable of the electrode power supply fits to the controller card installed at the computer e Optional Loudspeaker connector encoded green Connectors on most cables ensure error proof fitting Secure connectors with screws by fastening the screws 3 6 Setup of the power supply Connect the power supply to the sockets at the back of the electrophoresis chamber using the electrode power cable The red banana plug corresponds to the anode the black to the cathode Theoretically any power supply providing 250V 400 is sufficient for virtually all possible applications but we do explicitly not r
39. ol gilt nur in Staaten des EWR EWR Europ ischer Wirtschaftsraum umfasst die EU Mitgliedsstaaten sowie Norwegen Island und Liechtenstein Cet symbol conteneur d chets barr d une croix signifie que le produit en fin de vie doit tre retourn un des systemes de collecte mis a la disposition des utilisateurs finaux en cons quence des r gulations par la loi de votre pays Pour des information additionel nous Vous demandons de contacter votre distributeur Cet symbole s pplique uniquement aux pays de Espace conomique europ en qui regroupe les Etats membres de l UE et la Norv ge Islande et le Liechtenstein Instruction Manual Rotaphor 37 Biometra An Analytik Jena Company 12 Warranty This Biometra instrument has been carefully build inspected and quality controlled before dispatch Hereby Biometra warrants that this instrument conforms to the specifications given in this manual This warranty covers defects in materials or workmanship as described under the following conditions This warranty is valid for 24 months from date of shipment to the customer from Biometra This warranty will not be extended to a third party without a written agreement of Biometra This warranty covers only the instrument and all original accessories delivered with the instrument This warranty is valid only if the instrument is operated as described in the manual Biometra will repair or replace each par
40. specific lot of agarose Reaction conditions and times should be empirically determined However overnight digestion with clean reagents typically produces good results Other sources of agarose may also be tested to boost restriction enzyme activity Instruction Manual Rotaphor 27 An Analytik Jena Company 7 Operating 7 1 Equipment Rotaphor System 6 0 Thermostatic circulator or suitable other cooling option Sample mould delivered with the Rotaphor System 6 0 Gel casting tray and comb delivered with the Rotaphor System 6 0 7 2 Setting up the electrophoresis chamber Open the electrophoresis chamber and take out the gel support tray Pour 2 4 of the selected buffer into the electrophoresis chamber and switch on the computer if not already done Start the thermostatic circulator and allow the apparatus to cool to the desired temperature as displayed in the main window The optimum temperature range for most applications is 10 C to 15 C see below IMPORTANT Do not use more than 2 4 of electrophoresis buffer 7 3 Precautions There are some rules to be born in mind whenever using the Rotaphor System 7 3 4 Voltage The Rotaphor System is able to withstand a maximum voltage of 255 Volts Voltages higher than this will activate the fuse at the back of the controller and thereby disconnect the electrodes from voltage In addition it is possible to damage the Rotaphor System by applying voltages hi
41. t which is returned and found to be defective This warranty does not apply to wear from normal use failure to follow operating instructions negligence or to parts altered or abused 38 Instruction Manual Rotaphor Biometra An Analytik Jena Company 13 Glossary Corner Insulators Plastic pieces that prevent the buffer to fill the corners of the electrophoresis chamber They are necessary to make the homogeneity of the electric field independent of the rotor angle Duration Time until the end of electrophoresis Rotor V2 1 Rotatable field adjusted electrode array Interval Time between two rotor movements in other designs described as pulse time kb Kilo bases size of DNA molecules PFGE Pulsed Field Gel Electrophoresis Rotor Angle Angle between the two opposite positions of the electrodes Spirit Level Instrument to check whether a device takes up a correct horizontal position Startposition Position of rotor where the light barrier is interrupted Instruction Manual Rotaphor 39 Biometra An Analytik Jena Company 14 Trouble Shooting 14 1 Rotaphor does not function g 40 No action at all The pilot lamp does not light green when computer is turned on There is no power in your outlet Try another outlet Mains cable is not connected to the rear of the controller Check connection Mains cable is defective Replace with new power cable Computer works but chamber shows no reaction Check that all con
42. tandard agarose electrophoresis Mechanical shear forces are present in liquid phase samples and genomic DNA molecules above 500 kb are mechanically unstable and will fragment easily if stirred or pipetted To prepare and preserve intact chromosomal molecules for electrophoretic analysis a solid phase DNA preparation technique termed agarose plugs is typically used This protocol provides a basic starting point for Escherichia coli Saccharomyces cerevisiae and mammalian cells Following DNA isolation chromosomal DNA is typically digested or fingerprinted using restriction enzymes Many protocols have been developed over the years for restriction digestion of genomic DNA in agarose plugs The average pore size in an agarose matrix is typically quite large 100 200 nm thus allowing diffusion of enzymes and cofactors However to accelerate this process agarose plugs are made smaller either during preparation by making agarose beads or by mincing a large and fully prepared agarose plug into smaller pieces agarose chops It has been shown that the process of mincing and forming chops does not break DNA and this procedure is included adapted from Herschleb et al Nature Protocols Vol 2 No 3 677 684 2007 6 1 Preparation of agarose plugs Please note that the sample preparation will take 2 3 days excluding culture growth 1 Determine the shape and volume of the desired plugs Ideally the plugs should correspond to the dimensions
43. th some hot 1 agarose Now put the corner insulators over the distance pillars and press them down until they sit on the gel support tray see below The corner insulators are held in place by a plastic pin which has to be on the lower side Adhesion pulls up the agarose solution at the casting frame and at the comb This causes an uneven gel surface Especially after filling and capping the slots the 30 Instruction Manual Rotaphor An Analytik Jena Company buffer circulation above the gel is blocked by this agarose rampart Therefore cut away the agarose rampart with a scalpel to allow free buffer circulation Rotor Gel Distance Buffer circulation Corner pillar slot Insulator Positioning hole Positioning Pin Mount the 4 corner insulators as shown in the right part of the figure There should be no space between gel support tray and the corner insulators 3 7 Loading the electrophoresis chamber After the buffer has reached the desired temperature remove the safety lid This action prevents all current flow to the electrodes as well as to the motor Place the gel support tray with the gel and the corner insulators into the electrophoresis chamber and lower it carefully until the tray rests firmly on the inner edges of the tank The sample wells have to be in the back of the chamber near the electrical connectors This will ensure the nucleic acids migrate in the correct direction once the power suppl
44. ubate an additional two nights in NDSK at 50 C Mammalian cells sample preparation Pellet cells by centrifuging at 1 000g for 15 min at 4 C CRITICAL STEP Use a swinging bucket rotor to prevent premature lysis Discard the supernatant and wash the cells in 1 x PBS twice spin down the cells at 1 000g for 15 minutes at 4 C after each wash keeping the samples on ice during washes Count the cells using any reliable method CRITICAL STEP When resuspending pellets after the final wash and or count keep in mind that this solution will be diluted 1 1 into agarose Calculate cell concentration and resuspension volume accordingly Heat a 1 2 solution of LMP agarose in 1 x PBS and equilibrate to 42 50 C waterbath This solution can be held until ready for use Mix the cells and agarose together Mix the cells with an equal amount of agarose CRITICAL STEP Warm the cell suspension briefly in a 37 C waterbath before mixing with agarose When mixing take care to pipette thoroughly but slowly to avoid damaging the cells and shearing the DNA Keep the agarose cell suspension in a beaker of warm water on the benchtop to avoid premature gelation Pipette the agarose cell suspension into a casting mould taking care to avoid air bubbles Allow the moulds to set at 4 C until the agarose has gelled After solidification of the low melting point agarose plugs 15 min remove the tape and push the blocks out of the mould by using the plexigl
45. uced every pulse resulting in complete disappearance of the real band if the voltage is too high for the separation problem Especially if the size of the molecules becomes 4 mbp the voltage needs to be reduced to very low values 40V This leads to very long durations and limits therefore the upper size of DNA molecules that may be separated by PFGE 5 Temperature 20 The temperature has a relatively small influence on the separation Only the duration needs to be increased with lower temperatures If the temperature is lowered from 13 to 8 C the molecules move about 30 96 less down the gel in the same time Since the non separated molecules move also faster at high temperatures the upper part of the gel is wasted at elevated temperatures Additionally high temperatures lead to blurred bands Instruction Manual Rotaphor Biometra An Analytik Jena Company 5 Materials and methods Please note that not all of the listed reagents and buffers needed for an experiment Please read carefully the instructions in chapter 6 to decide which reagents and buffers have to be prepared 5 1 11 Reagents e 0 05 M EDTA pH 7 5 e 0 125 EDTA pH 7 5 5i Mercaptoethanol 0 5M EDTA pH 9 5 e PBS buffer e BSA acetylated if required for restriction enzyme e Triton X 100 membrane research grade e Restriction enzyme and corresponding 10x reaction buffer e Standard LE or high gelling temperature HGT agarose for standard gels e LM
46. ur during this testing Gel casting frame 20 x 20 cm frame Comb with 18 wells Four locating screws Computer Computer with pre installed Windows operating system Rotorgene software and controller card without monitor Power cord Manuals of the computer components Windows licence Keyboard Mouse Optional 3 1 4 3 1 5 3 2 Speakers Power supply Electrode power cable Power cord Connection cable to the interface card Block former Insert mould with 20 holes block remover Unpack and check Unpack and carefully examine the instrument Remove all adhesive tape Report any damage to Biometra Do not attempt to operate this device if physical damage is present Please keep the original packing material for return shipment in case of service issues Instruction Manual Rotaphor 3 3 Installation conditions e Place the Rotaphor System on a stable surface in a dry safe environment Placing the lid on an uneven surface or closing the lid with the gel casting frame mounted can damage the electrodes 3 4 Setup of the thermostatic circulator The thermostatic circulator can be ordered separately and is not part of the Rotaphor System The applied electrical field produces heat of 30 W that needs to be removed from the electrophoresis chamber We recommend a thermostatic circulator to cool the Rotaphor System electrophoresis chamber efficiently during the run Ideally it should contain at least 5
47. uration h 24 Temperature 6 E Interval sec 60 logarithmic to fio O Okb 5700kb 120h Okb S700kb 2401 Interval inverse sec Off Angle 120 linear to io Voltage V 180 logarithmic to fa 0 9 Agar Comments 0 3 x TBE New Parameter List Delete Parameter List Combined Lists Accept changes B SE Parameter Lists of Combined Lists e 20kb 900kb 42h 220 650 60h 2kb 1600kb 30h e e e z 10kb 1600kb 48h 30kb 2400kb 84h 50kb 2000kb 78h Hide Window New Combined List S me Assign Image Remove Image Figure 4 Rotaphor Software parameter editor To optimise the separation of samples it might be necessary to refine a parameter list and to program an individual protocol e Press New Parameter List Enter a name for the new parameter list in the field Name of List e Enter parameters for o Duration h 1 500 h o Temperature C 1 25 C o Interval sec 1 10000 sec o Angle 0 90 91 160 Voltage V 1 255V e For the interval angle and voltage two parameters each have to be set and the mode to be selected The mode defines the course with that the instrument changes between the set parameters If two identical values are programmed for a parameter the mode will be set to constant If two different values are programmed the mode can be set to linear to or
48. y is switched on and electrophoresis has commenced The gel should be covered by about 5 mm depth of buffer when the corner insulators are in place Any additional buffer required should be added at this point Be sure however that the buffer level does not reach the rotor electronics Place the safety lid on the lower part of the electrophoresis chamber and take care that all plugs fit into their respective sockets Now the lid is safely interlocked Wait for 5 to 20 minutes until the temperature of the gel is approximately equal to that of the electrophoresis buffer 3 8 Start and termination of electrophoresis After setting the parameters and pressing the Start Electrophoresis button the anode and cathode can easily be distinguished at least when TBE is employed as electrophoresis buffer because more bubbles develop at the cathode than at the anode DNA molecules move towards the anode therefore the anode should be the electrode at the front The electronics of the Rotaphor System interrupts current flow when the polarity is wrong The electrophoresis is terminated automatically but it might be interrupted by pressing the Interrupt Electrophoresis Instruction Manual Rotaphor 31 An Analytik Jena Company button at any time Remove the safety lid and the corner insulators Then lift the gel support tray out of the electrophoresis chamber Cut off the gel s feet with a scalpel and slide it gently from its support
49. ze Different amounts of DNA may lead to very different velocity of the same DNA size This effect influences even adjacent lanes The more DNA is applied the slower it moves Very blurred bands and or areas where no DNA can be detected Poor circulation of electrophoresis buffer Buffer pump is not working see above Inlet of the pump is blocked see above The gel surface is very uneven and blocks the buffer circulation above the gel The chamber is used the first time or it was stored longer than 2 weeks This phenomenon is seen sometimes To prevent this problem fill the chamber with desired buffer insert tray close lid and cool to 12 C Incubate for 1 hour change the buffer and start your electrophoresis Instruction Manual Rotaphor
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