ImaGo user manual. - Isogen Life Science
Contents
1. Username 8 characters This name is printed in the left bottom on the printout and data dumped image for identification purpose Normally this is the name of the department or the laboratory Password 8 characters This function permits the input of an extra password to be used to enter the setup The factory password BLS remains active Show overexposed pixels on off If quantification of bands or spots is required they may not be overexposed The highest concentration lightest band or spot should be within the dynamic range of the camera If this parameter is set to on by pressening the Enter key than all pixels which have an intensity above 99 5 will start blinking image should be frozen 18 The blinking is an indication for the user only and is not visible on the printout and or data dump Set auto exp image area Selecting this parameter followed by Enter will show the last image in memory with a rectangle super imposed in white With the keys you can change the size of this rectangle The active area inside the rectangle will be the part of the image which is used to automatically calculate the exposure time See Auto exposure function If the area is adjusted press Func to return to main setup screen JPEG quality 5 99 The data dump is the transfer of an image via the RS 232C interface For saving transfer time the image is compressed The compression used by th2. Available illumination cassettes Wavelength nm 256 302 As standard supplied 365 460 520 ImaVex 8 user definable wavelengths in the range of 400 700 nm Dual wavelength excitation Permits detection of fluorophores with a stokeshift gt 15 nm Available Top illumination Wavelength nm 256 302 365 450 3353 Data archiving Image archieving can be carried out by using the ImaComm archiving software This is a standard supplied package and should be installed on a Windows 95 98 NT or 2000 platform Installation in done by starting setup from disk one Follow the instructions on the screen during the installation Fig 1 shows the normal screen if the program is started Note that this software will run in background so it will not prevent you working on your PC Fig 2 Shows the setup which should be filled in to ensure the images are stored in the right format in the right directory Additional information is given on the screen C imagodatasS edecoversiel Fig 2 24 Fig 3 shows the communication parameters All should be according these setting The only exception is the communication port number that should be the same as the one on which the ImaGo is connected to Only change this if needed Fig 3 25 Applications Staining of ds and ss DNA and RNA in gels Fluorescent Dye s applicable for the ImaGo are Ethidium Bromide SYBR Green SYBR Gold Technical
3. Unique advantages of chemifluorescence are that imaging a fluorescent blot on a ImaGo fluorescence imaging system is nondestructive requires no film exposure and takes only a few seconds If an initial image is either weak or saturated adjust the sensitivity of the detection system to maximize the signal over background and then reimage or continue incubation with the substrate to develop additional signal With nondestructive imaging and adjustable sensitivity you can achieve the best results possible for each experiment 4 Stability In fluorescence light emission occurs only during exposure to the excitation light i e within approximately 10 nsec of absorption but a single fluorochrome molecule 35 can be excited repeatedly and can emit light each time Many fluorochromes are stable for a long time Fluorescent probes can be kept for many months and a fluorescent blot can be stored and reimaged after several weeks with little decay in signal In contrast a chemiluminescent molecule is autonomously luminous No excitation light is required for chemiluminescence however the luminescence peaks within minutes of substrate addition and decays over just a few hours 5 Limits of detection For most blotting applications the limit of detection of chemifluorescence is equal to that of chemiluminescence and is better than that of colorimetric detection 6 Established protocols Chemifluorescence uses the same labeling protocols a
4. analysis Store dye solution covered with aluminum foil to prevent bleaching of fluorophore by overhead lighting Stain solution can be re used when stored at 4 degrees C but must be strengthened by addition of fresh EtBr stock solution to compensate for that taken out of solution by the staining of previous gels Diluted stain solution will keep up to one month at 4 degrees C It is not advisable to do either restriction digests or blotting after EtBr staining CAUTION Use gloves due to mutagenicity Powder can be stored at room temperature but must be protected from light In aqueous form the stain should be stored at 4 degrees C protected from light Use gloves due to mutagenicity Experimental information for SYBR Gold Advantages More sensitive than EtBr Allows post stain processing of DNA e g restriction digest or Southern blotting Replaces silver staining and radioactive labeling in some applications e g SSCP Can be used with glyoxal and formaldehyde gels Quick quantitative analysis using the Intelligent Quantifier software SYBR gold can be used in the presence of urea glyoxal and formaldehyde No need to destain because of low background Allows excision of bands for cloning after heteroduplexing Excitation Maximum 300 nm Emission Maximum 537 nm 28 Post Staining Run gel Use centrifuge to spin stock solution to bottom of tube Dilute Pre Staining Pre casting Binding SYBR Gold to a final dil
5. applications Gel Shift Assay AFLP Amplified Fragment Length Polymorphisms Heteroduplexing MVR Multiple Variable Repeats Purity checking of DNA prior to sequencing RT PCR quantitation Reverse Transcriptase PCR STR Short Tandem Repeats mRNA differential display RNA quantitation SSCP Single Stranded Conformational Polymorphisms DNA quantitation Technical Background RFLP The human genome contains a wide variety of DNA sequences that are present in multiple copies The exact number of copies of each repeated sequence varies with each individual RFLP is a technique for looking at the variation in the copy number of these repeats by cutting genomic DNA with specific restriction nucleases separating them on an agarose gel and then hybridizing probes specific for different repeated sequences The information obtained can then be used for identification purposes AFLP Amplified Fragment Length Polymorphism Similar to RFLP the difference is in the way the fragments are produced In RFLP regions of repetitive genomic DNA 26 are lit up with a probe after all the DNA has been cut into small pieces by a restriction enzyme In AFLP the fragments are produced by PCR amplification using unique sequences that flank the repetitive region Again since each one of us has a particular repetitive set of regions which contain a variable number of repeats individuals can be identified by the size of the different fragments The
6. is marked with this symbol refer to the instrument manuals to protect the instrument against damage WARNING A WARNING indicates a potentially hazardous situation which if not avoided could result in death or serious injury CAUTION A CAUTION indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Do not proceed beyond a WARNING or CAUTION notice until you understand the hazardous conditions and have taken the appropriate steps Note A Note provides additional information to aid the operator in obtaining optimal instrument performance Warning Label Warning labels are attached at several locations on this instrument Do not remove deface or damage the warning labels If a warning label peels off the instrument or becomes illegible contact your local ImaGo distributor for a replacement label Warning for FUSE Only use fuses of the designated rating to protect both operator and instrument from fire and other hazards The warning labels that pertain to fuse ratings are located on the back panel of the instrument and inside near the fuses Fuse 14 slow Pmax TS 230V4C 50Hz Disconnect from mains before opening Warning Installation Unpacking and placing the instrument 1 Unpack the instrument by opening the box and take out all separate boxes 2 Takeout the instrument of the box and directly place it on top of
7. switched on the ImaGo logo will appear To continue press Func Enter or print If no key is pressed the software willautomatically after 10 seconds continue with normal operation Keyboard layout Step 1 Open the drawer and place your sample on the ilumination cassette Step 2 Close the drawer and switch on the appropriate illumination source Step 3 Adjust the exposure time for a good visable image zoomed out Step 4 Position your image with the arrow keys in such a way it fills the whole screen This in combination with the ZOOM keys Step 5 Press Enter key to freeze the image Step 6 If wanted adjust image and make a printout by pressing Print Electronic image positioning One of the unique features of the ImaGo is the electronic image positioning This can simply be accompliced using the arrow keys It means you only have to take care your sample is alligned in horizontal position and you do not have to take care where it is on the illumination cassette It is also possible to image the sides of the sample and zoom in for a detailed view The sample remains at its initial place only the viewing position is chanced 10 Electronic optical zooming This feature allows you to zoom in and zoom out with full optical resolution The maximum zooming range is 6 x This means a viewing range from 4 7 x 3 5 cm to 28 x 20 cm Additional operating information After placing your sample on the illuminat
8. than with EtBr alone but inferior to pure SYBR Gold staining Avoid use of bind silane with SYBR Gold on polyacrylamide gels it is preferable to use a Gel Slick type product on the opposite plate Staining of extra large sandwich gels can be easily accomplished by laying the gel sandwich onto paper towels having cleaned the glass plates well first Remove top glass plate then pipette enough 1 10 000 SYBR Gold stain onto gel to cover completely Spread evenly with Pasteur pipette and leave protected from light for 10 60 minutes If required SYBR Gold can be removed from dsDNA by simple ethanol precipitation Bring solution up to 10Mm NaCl add 2 5 volumes 95 ethanol incubate 20 minutes on ice centrifuge for 10 min at 4C Storage In DMSO in plastic protected from light Stock solution kept at 20 degrees C is stable for six to twelve months Diluted reagent kept at 4 degrees C is stable for three to four days Handling Treat as potential mutagen as data is unavailable Vendor Molecular Probes Inc www probes com Staining of proteins in 1 D amp 2 D Gels Fluorescent and non fluorescent dye s applicable for the ImaGo SYPRO Orange Coomassie blue copper zinc and silver stain Technical applications 1 D protein electroforesis 2 D protein electroforesis Proteomic analysis Western blot analysis lt 3 Experimental information for SYPRO Orange Advantages Simple quick staining protocol As sensiti
9. Attophos Always image immediately Some blots can develop within minutes Use of water between imaging tableglass and plastic bag reduces optical refraction and results in a clearer image Use Mylar rather than Saran wrap as a cover It is far less fluorescent Covered refrigerated solution can be used up to one month Use of containers and tips cleaned by sterilizing or by rinsing with 0 01M HCl or boiling water reduces background resulting from ambient alkaline phosphatase Autoclaving of blocking solution and filtering of reagents can decrease background from ambient alkaline phosphatase Use of powder free gloves is important as the powder fluoresces siTe Trouble Shooting No operation at all Check fuses at the main entry If needed replace both for 110V 2A or 230V 1A Power LED on but no image Check if TOP light operates If still no image check diaphragm and zoom out fully If still no image check fuses on main board Zooming not functioning Check functioning of zooming at lens itself If zoom motor turns check rubber wheel No image positioning possible in vertical direction Check if camera is moving when cursors are pressed If not call service No image positioning possible in horizontal direction Check if moving mirror turn very little if cursors are pressed If not call service Image is not sharp over the entire zooming range Lens is misaligned Call service for
10. Protein detection For protein detection using the Vistra ECF Western Blotting Kit on blots or microplates the primary antibody is recognized by a secondary antibody that is conjugated to fluorescein Amplify the signal by incubating the blot with the anti fluorescein tertiary antibody conjugated to alkaline phosphatase followed by the substrate The substrate is cleaved by alkaline phosphatase to yield a fluorescent signal The chemifluorescence advantage 1 Specificity and versatility Like chemiluminescence chemifluorescence is based on the affinity binding of an enzyme conjugated probe to the target molecule Alkaline phosphatase is readily available conjugated to antibodies that target a wide variety of haptens or to streptavidin The use of antibodies ensures specific detection while allowing great versatility in choice of target molecules for both protein and nucleic acid detection 2 Signal amplification Under the proper conditions enzymes continue to convert substrate to product as long as the substrate is available In chemifluorescence each alkaline phosphatase conjugate can generate many fluorochrome molecules continually increasing the signal at any given target For many samples only a few seconds of incubation with the substrate yields sufficient signal However blots can be incubated for as long as 24 hours to maximize the signal from low abundance targets 3 Nondestructive imaging and adjustable sensitivity
11. The image will be processed according to the latest used parameters Pressing the Func key will change to the next function The Print key is only active if the image is frozen oe Ge After selecting the desired function the monitor will indicate the chosen function in the statusbar bottom of the screen Note To escape from any point in the menus to the acquisition mode press Func key and then Enter key while keeping the Func key pressed Not valid for setup mode Note Ifthe screen is dark due to the screen saver press Func or Enter to restore the screen no key function operation is performed wis Reference chart and function description Function Key range Enter key function Manual Exposure mode live 0 001 20 sec Freeze Live Manual Exposure mode frozen Auto Exposure Start detection Data Dump image Contrast 9 to 9 Invert toggle Brightness 0 to 100 Hi Lo Magnify 1x to 4x Reset to defaults Setup Enter setup Note To return from one of the above functions to image acquisition live press Func and Enter keys simultaniously Manual Exposure mode live The exposure time is the time used to acquire the image from your sample If faint bands or spots are not visable raise the exposure time If the image becomes saturated to white for fluorescence lower the exposure time The image is updated after every exposure The exposure time can be set from 0 001 to 20 sec F
12. User manual The new Multi purpose Compact Imaging System 1 10 2001 Software version 2 03 B amp L Systems Industrieweg 68 3606 AS Maarssen 31 346 550556 the Netherlands Index Introduction Specifications Safety Considerations Installation Basic operation Electronic image positioning Electronic optical zooming Additional operating information Reference chart and function description Auto Exposure Data dump Contrast Brightness Magnify Setup Username Password Show overexposed pixels Set auto exp image JPEG quality Dump mode Last printout Baudrate 10 10 11 11 13 14 14 15 16 17 18 18 18 18 19 19 19 19 20 Printout with dump UV Top Machine name Exit the setup Matching monitor and printer Changing the print format Changing the emission filters Produce sharp images Standard available emission filters Available illumination cassettes Available Top illumination Data archiving Applications Staining of ds and ss DNA and RNA in gels Experimental information for Ethidium Bromide Useful tips for Ethidium Bromide Experimental information for SYBR Gold Useful tips for SYBR Gold Staining of proteins in 1 D amp 2 D Gels Experimental information for SYPRO Orange Useful tips for SYPRO Orange Detection of proteins or DNA on membranes with ECF Protein detection The chemifluorescence advantage Experimental information for Attophos ECF s
13. advantage of AFLP over RFLP is that less material is needed to identify the individual SSCP Single Stranded Conformational Polymorphism Unlike Southern blot analysis SSCP relies on the migration characteristics of single stranded DNA which is very dynamic in its kinetic action This structure allows it to either migrate faster or slower through a gel Some researchers rely on this technique to give them information about single base substitutions that are known to be associated with disease Experimental information for Ethidium Bromide Advantages Easy inexpensive to use Detects 2 ng band dsDNA in agarose or acrylamide including denaturing gels Detects 100 ng band RNA in non denaturing agarose gels Greater dynamic range on the ImaGo system than Polaroid film Rapid quantitative analysis using the Intelligent Quantifier software Excitation Maximum 302nm Emission Maximum 615nm Post Staining Run gel Stain for at least 20 minutes in 0 5 micrograms ml EtBr in water TE or TBE on a shaker Destain for 20 minutes in water Pre casting Cast gel using a final concentration of 0 5 micrograms ml EtBr Run gel using 0 5 micrograms ml EtBr in running buffer Destain for 20 minutes in water optional TE TAE or water can be used to make up the 0 5 micrograms ml EtBr stock solution Binding EtBr is a non covalent intercalating dye Detection Limits Detection of ssDNA and RNA is less sensitive than for dsDNA Exact figures for ssDNA are
14. e ImaGo is JPEG As a standard 50 is chosen which is right for almost all applications Increasing this value will increase the file size and transfer time but decreases the image alteration It is not recommended to change this parameter If the new value is entered by using the keys press Func to return to main setup screen Dump mode ASCII binary If ASCII is selected the data transfer is in full readable ASCII characters else the binary function is selected and the image data in binairy format The output file has the format Image output starts with Followed by the Image number 7 ASCII characters Followed by a Image data ASCII or binary End of file character Example 000025 filedata EOF The ImaComm software will store these data in different image formats again on the PC See for further explanation the chapter ImaComm data archeiving Last printout 0 999999 The image number is updated automatically after every printout and or data dump If a upload of new firmware is carried out the image number is set to 0 It is possible to enter the old image number The highest number is 999999 Overflow will result in reset to 0 Baudrate 9600 19200 38400 57600 115200 This number indicates the transmission speed of the RS 232C port of the ImaGo Normally it is set to 115200 If the network transfer unit is used it is prefered to use 57600 The maximu
15. e changed Magnify Digital magnification can be selected in the range from 1 to 4 times Magnification is always calculated from the image centre Original 3 times magnified Exp 00 120 Con O0 Hi 100 Lo 0 Mag 1 Exp 00 120 Con 0 Hi 100 Lo 0 Mag 3 Customer Normal UU 0ff WL On_ 000013 InmaGo Customer Normal UV Off WL On 000012 ImaGo Reset to defaults within magnify function The Enter key will reset all the image process functions to the default values These are defined in the setup function The exposure time is also set to the default but this is only visible at the first new live image a 17 Setup In the setup function system parameters and default values can be stored The parameters are Exposure time Contrast Inverse normal image Brightnes Hi and Lo Magnification Password Note The values stored to defaults are the values active at the moment you enter the setup function Note Shorthand information is available in the status bar at the bottom of the screen Select the function and press Enter The image disappears and the password BLS can be entered If an extra password is defined in the setup this one can be used as will During the input of the password the characters are readable Characters can be selected with the and keys followed by the Enter key Pressing the Enter key can skip unused characters Choose one of the 11 parameters by pressing the or keys
16. e safety rules EN 61326 1 classe B and EN 61010 It is therefore that we certify these products with the CE marking Products ImaGo 500 MZ ImaGo 1200 MZ All supplied illumination cassettes 15 April 2000 B amp L Systems The Netherlands H Beijersbergen van Henegouwen Director 40
17. er is stable for one year when stored at 4 degrees C Method Use 1mM ECF Substrate dissolved in buffer or use kitconcentration For all procedures including Southerns westerns and northerns use clean containers pipette tips etc and pipet ECF Substrate onto a 36 overhead projector sheet and lay blot face down onto solution Be sure to coat blot completely and evenly with substrate Incubate as needed then transfer blot face down to imaging surface Always image immediately in case of rapid signal development then every 15 minutes up to one hour then every hour leaving overnight if necessary For western blots on PVDF membranes blots can be scanned either wet or dry Binding ECF Substrate is dephosphorylated to the fluorescent product which remains in the region in which it is produced It is believed to adhere to the membrane by charge interactions Detection Limits Variable depending on the application 10 M of product can be detected in a microplate well Linear Range 1 3 orders of magnitude highly variable depending on application Emission Filters Attophos filter Storage In clean glass or plastic refrigerated and protected from light In buffer 2 4M DEA pH 10 0 0 23mM MgCl2 Diluted reagent at 4 degrees C keeps up to one week Handling Use powder free gloves to protect ECF Substrate from naturally occurring alkaline phosphatase on hands Vendor Amersham Life Science Inc Useful tips for
18. ilable Stock solution contains DMSO and should be handled with double gloves Vendor Molecular Probes Inc www probes com Flea Bide etek Fluen eneon Useful tips for SYPRO Orange Ensure glass plates and staining containers are completely free of grease and fingerprints Use powder free gloves to handle gels Mix staining solution fresh immediately before using Shake staining solution vigorously before adding it to the gel Staining in acetic acid significantly impairs immunodetection For western blotting stain in transfer buffer sensitivity may be slightly diminished Staining solution can be reused effectively at least three times When staining native gels soak gels in 0 1 SDS in either 7 5 acetic acid or water for 30 60 min prior to staining Sensitivity will be diminished in native gels Methanol can adversely affect staining To eliminate methanol soak gel in 7 5 acetic acid for 1 2 days prior to staining When staining acidic proteins increase the concentration of SDS in the gel and buffers to 0 2 33s Detection of proteins or DNA on membranes with ECF Chemifluorescent substrates applicable for the ImaGo ECF Substrate AttoPhos Technical applications Colony plaque screening Dot slot blots Human DNA quantitation forensics RFLP VNTR Southern blotting Western blotting Colony Hybridization Plaque Lifts Technical Background Chemifluorescence is the enzymatic conversio
19. ilable to change the black and white distribution as indicated in figure 1 The Enter key will toggle between normal and inverse image Inverted image Contrast curves fig 1 Contrast setting amste Ke tae mo gupes a it ens ep a 5 5 ea Exp 00 120 Con 0 Hi 100 Lo 0 Mag 1 Customer Invert UV Off WL On 000011 Two examples of the contrast setting Contrast 9 Contrast 9 m Exp 00 120 Con 9 Hi 100 Lo 07 Mag 1 Exp 00 120 Con 9 Hi 100 Lo 07 Mag 1 Customer Normal UU Off WL On 000005 Customer Normal UU Off WL On 000004 _ _ImaGo 15 Brightness The brightness settings contains two parameters The upper and lower limits are called Hi and Lo Toggle between these two parameters is done by pressing the Enter key The value is a percentage of the total image black to white range e g black is 0 and full white is 100 Example Lo 25 and Hi 85 This means that pixel intensities below 25 will remain black The pixel intensities above 85 will all be at maximal white So the remaining 25 to 85 will be stretched to a visable range from 0 to 100 Increasing the Lo value will subtract background Decreasing the Hi value will show faint bands or spot more bright 100 Original New pixel intensity Pixel streched to a range from Intensity 0 to 100 0 Note It may take up to 1 5 seconds before the image is fully processed if the Lo or Hi values ar
20. ion cassette you have to switch on the source This can be the TOP light standard is white or the BOTTOM light illumination cassette The keys are named accordingly A LED next to the key will illuminate if the illumination is switched on If the drawer is not closed the bottom light will be off even if the keyboard LED indicates it is on To bypass this safety switch simply pull the white knob at the left top of the drawer hole This function enables you to cut fragments with the drawer opened Note If the illumination source is not properly pressed towards the back of the drawer hear the click it will not operate as well WARNING UV radiation can be harmful Wear eye protection if used in this way In normal acquisition mode no functions are available You can only change the exposure time with the and keys Image positioning zoom and illumination keys remain active The live image is always an unprocessed picture This to be sure that the widest dynamic range can be used Image positioning can easily be done with the arrow keys If you would like to zoom a specific part of your sample you can do so with the ZOOM IN and ZOOM OUT keys Note The bottom illumination cassette will switch off automatically after 10 min If the image is frozen the light source can be switched off This is advisable to prevent the sample from denaturation The information on the monitor will change and indicate the active function
21. m RS 232C cable length at 115200 baud is 5 meter Lowering the baudrate will increase the maximum cable length used for the image transfer Printout with dump on off If on a printout will be generated at the same time a data dump is made This ensures that PC data and printout are identical UV Top Not installed installed If the UV toplight option is installed it can be switched on by selecting both the Top and bottom light In this case UV Top will be printed dumped as illumination source Machine name ImaGo LumaGo The ImaGo can optionally be equipped with a luminescence unit In this case the ImaGo will be renamed as LumaGo It only effects the name on the printout datadump At the bottom the setup screen a test bar is shown which changes from black to white and visa versa The can be used to match the printer and monitor 20 Exit the setup To exit the setup press Func From here you have to chose Yes or No Yes means that all values are permanantly stored in the ImaGo After switch off and on the ImaGo the values will still be active If answered with no the values are valid but only until the ImaGo is switch off O Matching monitor and printer If you have entered the setup mode you will see a testbar at the bottom of the monitor It starts at 0 black and goes linear to 100 white and back again To be sure that the printer is producing the same printout as
22. n of a fluorogenic substrate to a fluorescent product Fluorogenic compounds non fluorescent or weakly fluorescent substances that can be converted into fluorescent products are available for use with a wide variety of enzymes ECF reagent kits Amersham Life Science simplify chemifluorescence for membrane based protein and nucleic acid detection The kits use alkaline phosphatase which cleaves a phosphate group from a fluorogenic substrate to yield a highly fluorescent product Fluorescence Substrate 550 570nm 9 Excitation t r a Fluorescent y product ar A A wv Alkaline Phosphate phosphatase group Alkaline phosphatase and horseradish peroxidase are currently used interchangeably in most colorimetric and chemiluminescent membrane detection assays including southern northern western and slot or dot blotting Chemifluorescence can be used in place of any standard chemiluminescent protocol simply by using an alkaline phosphatase conjugate and the chemifluorescent substrate Standard buffers and incubations remain unchanged After a short incubation with the substrate image the blot on the ImaGo The light source in the instrument excites the fluorescent a4 product which emits light in a band centered around 560 nm The instrument detects this fluorescent signal and creates a digital image of your sample Genomic Solutions Intelligent Quantifier TM software allows rapid and accurate quantitation of the results
23. not currently available Sensitivity will always be better in acrylamide gels than in agarose gels because acrylamide gels have lower background Sensitivity 2 ng dsDNA band depending on fragment size 100 ng RNA band in non denaturing gels Larger fragments will bind more EtBr Linear Range 1 5 orders of magnitude Emission Filters Ethidium Bromide filter Absorbance Fluorescence emission 400 450 500 550 600 650 700 750 Wavelength nm Useful tips for Ethidium Bromide Binding efficiency and sensitivity of EtBr with ssDNA and RNA is less than with dsDNA EtBr and SYBR Green I compete for DNA binding Staining first with EtBr then with SYBR Green I will give better results than with EtBr alone but inferior to pure SYBR Green I staining For use in agarose and polyacrylamide gels To stain denaturing gels wash gel to remove urea before staining Longer stain times do not improve sensitivity However when dealing with highly concentrated tightly packed bands in a high percentage gel allow more time for the EtBr to penetrate further into the bands to improve visualization Staining and or destaining for less than 20 minutes is not advised Destaining longer than 20 minutes can decrease band signal strength Pre casting gels with EtBr can save time after electrophoresis but may also alter migration of fragments EtBr migration causes non uniform background staining in pre cast gels Post staining should be used for quantitative
24. or Ethidium Bromide stained gels an often used exposure time is 0 5 sec Pressing Enter will freeze the image and all other functions will be available The statusbar at the bottom of the image will change and show all parameters used including the functionality of the active control keys Note In this mode the Print key is not active Manual Exposure mode frozen From this point on you can print an image just by pressing the Print key This can be done from all functions except during setup Pressing Enter in this mode returns to the image acquisition mode live again 3 Auto Exposure The Auto Exposure function enables you to detect the optimal exposure time The function will automatically adjust the exposuretime based upon the image rectangle defined in the setup see setup function After detection the ImaGo will switch over to manual exposure mode live This to have the possibility to readjust the exposure time manually Data dump If selected pressing the Enter key will cause the image to be transferred to the connected Windows based computer Transfer is made over the RS 232C interface Optional a direct image transfer over UTP network is available In the setup function a selection can be made if a printout is made parallel to the data dump This ensures to have the image and printout exactly the same 14 Contrast This function will change the contrast of the image There are 18 different curves ava
25. osure time Contrast setting Image inverting Brightness Hi Lo Bottom illumination Electronic optical zooming Digital magnification Image sensing Reset to defaults Screensaver Printout Data output Filetype Used paper Image size std Sensitivity Monitor Main Outer dimensions Weight 0 001 to 20 sec Yes Yes 9 to 9 18 curve types Yes 0 to 100 Standard 302 nm 20 x 25 cm with automatic switch off 10 min 6x 1 4x ImaGo 500 DSP controlled CCD camera 748 x 655 pixels ImaGo 1200 DSP controlled CCD camera 1264 x 1024 pixels Yes After 10 minutes no keyboard activities On build in photoquality thermal printer with GLP information High speed RS232C and High resolution video optional TCP IP JPEG 1 100 K61B normal or K65HM glossy 20 x 25 cm to 3 4 x 4 2 cm lt 0 0005 Lux High resolution build in with dark clamping 210 to 240 Volt AC 50 60 Hz 180 Watt max Installation Cat II 520 x 380 x 500 W H D 49 kg Safety Considerations To ensure operation safety this instrument must be operated correctly and maintained regularly Carefully read to fully understand all safety precautions in this manual before operating the instrument This manual denotes precautions against actions that can result in hazardous situations or equipment damage by using the signal words WARNING CAUTION and Note A N Instruction manual symbol If the product
26. procedure Illumination sources are not functioning but LED s switches on off Check if illumination cassette is properly inserted in the drawer Check fuses at the mainboard ImaGo seems to operate normally but no sample results are obtained even these can be observed by eye Check emission filter on lens Fully zoom out and check again Warranty The ImaGo is supplied with a full year warranty Warranty does not cover the illumination cassette filter and lamps the top light source and the emission filter Warranty is void if instrument is serviced by unautherised personnel B amp L Systems can not take any responsibility for the functioning of the ImaGo or its safety if the instrument is not properly handled IRS Maintenance The ImaGo is designed to operate with a minimum of maintenance Despite the closed construction it may appear that the emission filter on top of the lens and sometimes the movable mirror above the lens has to be cleaned This is only due to the touching of these components by hand Cleaning can easily be done with ethanol water 50 50 Take care not to scratch the surfaces After cleaning take care that the lens is focussed properly WARNING AN Cleaning the lens should only be done with a soft optical cloth 39 Declaration of conformity B amp L Systems herewith confirm that the below mentionned products are fully tested and have been approved for all applicabl
27. s chemiluminescence A chemiluminescence system that already uses alkaline phosphatase can become a chemifluorescent system simply by switching to the chemifluorescent substrate For a system using horseradish peroxidase HRP just replace the HRP conjugate with an analogous alkaline phosphatase conjugate and incubate with the chemifluorescent substrate Standard buffers and incubations remain the same 7 Quantitation Chemiluminescence is linear over about a tenfold range In an analogous system chemifluorescence is linear over about a 50 fold range Experimental information for Attophos ECF substrate Advantages Comparable sensitivity to chemiluminescence Eliminates the need for dark room film and chemiluminescent screens Greater dynamic range than film allows visualization and quantitation of weak and dark bands in the same image Substrate for alkaline phosphatase Converts by enzymatic reaction to fluorescent product which emits light when excited by a suitable excitation light source Alkaline phosphatase linked secondary antibodies and ECF substrate can be used to amplify signal detection in a variety of microplate and membrane based applications such as southern northern western dot and slot blots No probe purification step necessary Probes stable at least six months at 20 degrees C Excitation Maximum 440 nm Emission Maximum 560 nm Buffer 2 4M diethanolamine DEA in water 0 23mM MgC12 pH 10 0 using NaOH Buff
28. the bench It is recommended to level the instrument because the sample can float on top off the illumination cassette This will result in an unsharp image 3 Take care that there is minimal 10cm distance between backside of the instrument and the wall It is advisable to keep some more space on the right hand side because of the better accessibility of the camera hatch WARNING The ImaGo must be connected to a main outlet 220VAC 50Hz with grounding 4 Unpack the illumination source standard 302 nm 5 Remove the drawer lock which prevent the drawer to open during transportation 6 Open the drawer and place the illumination cassette in the drawer and carefully but firmly press it against the backside of the drawer If the cassette is correctly inserted a click can be heard 7 Remove the transportation screw on the right hand side to unlock the camera driving system WARNING If locking the camera is needed for renewe transportation be carefull not to overtighten the transportation scrw It meight harm the camera unit 8 Open the printer front with the small bar on the left hand side of the printer 9 Insert the printer paper as indicated on the label inside 10 Close the printer front 11 Switch on the ImaGo with the main switch located at the back of the instrument 12 If needed adjust sharpness see Operating the ImaGo Your ImaGo is now ready for use Basic operation After the instrument is
29. ubstrate Useful tips for Attophos Trouble Shooting Warranty Maintenance 20 20 20 21 22 22 22 22 23 23 23 24 26 26 27 28 28 31 31 32 33 34 35 35 36 37 38 38 39 Declaration of conformity 40 This document is subject to confidential approach and information additional to the European standardized A form declaration None of this document s information may be copied or reproduced as a hole or in part in any way without written consent of B amp L Systems Introduction Congratulation with the purchase of your new ImaGo imaging system The ImaGo is a state of the art digital imaging system designed for instant photography computerized image investigation for gels blots and other samples Due to the unique optical design the ImaGo is extremely small and suitable for a wide variety of applications There is no need for manual positioning the sample after it has been put in the instrument because of the four positioning keys which permits the user to move the image in all directions An electronic optical zooming can magnify your image to accommodate all sample sizes The use of easy to change illumination cassettes allows multiple fluorophores to be excited There is no need to make any adjustments on the optical components On the photoquality printouts all active parameters are printed for GLP use Specifications Exposure time integrating Image freezing Automatic exp
30. ution of 1 10 000 in TE TBE or TAE pH 7 0 8 5 Leave gel to shake in dye solution for 10 30 minutes longer for thicker or higher percentage gels No destaining step necessary Incubate DNA with 1 5 000 SYBR gold for 15 minutes prior to electrophoresis Add loading dye after 15 minute incubation Preferred method for high percentage gels Dilute SYBR Gold to 1 10 000 into gel solution just prior to casting Add 1 10 000 dilution of SYBR Gold to running buffer SYBR Gold is thought to bind non covalently to the phosphate backbone of the DNA molecule Detection Limits Detection of ssDNA and RNA is less sensitive than for dsDNA Post staining Pre staining Linear Range Exact figures for ssDNA are not currently available Sensitivity will always be better in acrylamide gels than in agarose gels because acrylamide gels have lower background in acrylamide 100 pg dsDNA band depending on band size 1 3 ng ssDNA or RNA 10 20 ng synthetic 24 mer oligonucleotide 400 pg in 0 8 agarose 150 pg in 6 acrylamide 450 pg in 0 8 agarose 1 5 2 orders of magnitude Emission Filters SYBR gold filter 29 LAER KAA DNH Useful tips for SYBR Gold Use double gloved protection against DMSO stock solution Cover dye solution with aluminum foil to prevent bleaching of fluorophore by overhead lighting SYBR Gold and EtBr may compete for DNA binding Staining first with EtBr then with SYBR Gold will give better results
31. ve as silver staining 2ng band for most proteins Linear range from 2ng to at least 1 microgram Allows western transfer and immunodetection after staining Nonspecific fluorescent staining of denatured proteins in 1D and 2D SDS polyacrylamide gels Excitation Maximum 300 nm Emission Maximum 570 nm Binding Detection Limits Nonspecific SDS dependent binding to proteins As for all protein staining systems dye binding will depend on the amino acid composition of the proteins Proteins in native gels will exhibit more staining variability than proteins saturated with SDS 56ng band for most proteins in denaturing gels Run gel Dilute SYPRO Orange 1 5000 in 7 5 acetic acid enough to cover gel Shake vigorously Place gel and stain in a foil covered container and shake gently for 30 60 min Optional destain in 7 5 acetic acid for 10 15 min Rinse briefly in distilled water Alternative Protocol for Western Transfer Dilute SYPRO Orange 1 5000 in Tris Glycine Methanol transfer buffer Proceed with staining as above If necessary destain in transfer buffer Linear Range Emission Filters Storage 2 orders of magnitude SYPRO Orange filter Stock solution is stable for six months to one year stored at 20 degrees C and protected from light Staining reagent diluted in buffer or 7 5 acetic acid can be stored protected from light at 4 degrees C for at least three months ye Handling No toxicity data ava
32. what you see on the monitor WYSIWIG you press the Print key on the printer itself to produce a printout Normally the matching is done at the factory but over time readjustment may be necessary Refer to printer manual to change these parameters Printer factory settings Brightness 6 0 Contrast c 0 Printsize SN Gamma R5 Changing the print format The printer in the ImaGo can produce several printout formats The procedure is clearly written in the manual supplied with the printer Changing the emission filters To change the emission filter slide down the cover on the right hand side The lens will be visible Unscrew the filter while keeping the lens adjustment in its place Mount the new filter in reverse order Follow the next procedure to adjust the sharpness of the image Produce sharp images If image is not sharp than put a testjig on the illumination cassette and switch on the top light Take care that the diaphragm lowest ring of the lens is locked on 3 0 not 1 0 Adjust the brightness of the image by changing the exposure time Normally around 0 3 sec Fully zoom out and adjust the upper ring of the lens to get the best image distance After this fully zoom in and readjust fine adjustment 399 Standard available emission filters Wavelength nm BW 600 100 high sens As standard 530 40 e g Sybr Green 560 40 e g ECF Note Others in the range of 450 to 700nm on request
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