1 Date of Revision: April 16, 2012
Contents
1. 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 Et FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 PAGE VersaDoc Standard Operating Procedure DATE NAME amp EXTENSION SUPERVISOR SAMPLE DESCRIPTION CAMERA AND LENS TRAY USED F STOP OPTIMIZED PROBLEMS GEL OR BLOT AND USED NUMBER EXPOSURE TIME COMMENTS DYES TAGS USED 4000 amp ZOOM LENS WHITE TRAY 4000 8 FIXED LENS UPPER CL TRAY 5000 LOWER NO TRAY 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 Et FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 50002. 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 PAGE VersaDoc Standard Operating Procedure PAGE
3. F stop Time Notes seconds Protein gel stained White light with colorimetric Visible strong signal 5 6 or conversion screen d 1 0 1 to 5 0 ye coomassie expected higher required on lower silver tray Protein gel stained Fluorescent 5 6 or Place directly on with fluorescent dye excitation strong hi her 1 0 to 10 lower tra Sypro Ruby GFP signal expected 5 Ultraviolet Ethidium Bromide excitation 1 4 to 1 0 to 15 Place directly on DNA gel moderatly strong 5 6 i lower tray signal expected Chemiluminescent or enhanced Place on upper H t u Blot or Dot chemiluminescent 1 4 100 500 chemiluminescent weak signal tray expected These settings assuming the 4000 camera and fixed lens are installed The 4000 camera and the fixed lens are suitable for most applications however If the signal is very weak or the sample is too large it appears cropped in the image please speak to the Instrumentation Technician about using the 5000 camera or zoom lens DO NOT attempt to change the camera or lens yourself Unless the sample is larger than 8x8 cm in which case it should be placed on the lower tray 9 In the acquisition window under Step II select POSITION and manually adjust the position of the sample if needed note minimal adjustment is probably required as the image analysis software imagelab can be used to crop and rotate an image as needed 10 Select FOCUS and confirm that the sample is focused If
4. proper procedures Before using ANY hazardous materials make sure you understand the proper clean up procedure The Environmental Occupational Health and Safety Office 15 also available to provide guidance at ext 2874 See the WLU Laboratory Health and Safety Manual for further details All users must clean up any spills that occur in or around the VersaDoc working area including the plates inside VersaDoc hood the lab bench and the floor Although the plates White light conversion plate UV transilluminator tray Chemilluminescent tray are resistant to most chemicals they can be damaged if exposed to chemicals for a long time or by inappropriate cleaning NOTE If you are running DNA gels using Ethidium Bromide please do NOT touch any other parts of the instrument with contaminated gloves i e computer bench outside of instrument etc Please make sure you clean up all possible EtBr contamination thoroughly 6 WASTE DISPOSAL PROCEDURES All WHIMIS Department and Health and Safety guidelines must be followed for disposal of all chemicals used for analysis All sample and chemical disposal must be done in the user s lab NOT SR416 and disposal must conform to the guidelines set out by the EOHS Office See the WLU Laboratory Health and Safety Manual and the Biosafety Manual for more details 2 https www wlu ca forms 706 AccidentincidentinvestigationReportForm pdf 3 http www wlu ca documents 33337 Accident_Investig
5. User Guidelines Standard Operating Procedure BioRad VersaDoc Imaging System Date of Issue May 16 2011 evision 1 Date of Revision April 16 2012 VersaDoc Standard Operating Procedure ii TABLE OF CONTENTS DISCLAIMER costo o iii ACKNOWLEDGEMENT Soi E EA EA SEENA ees iv 1 INTRODUCTION ais Aaa 1 1 1 Purpose of the Standard Operating Procedure 1 1 2 Theoretical Background ooocoococonccnonconcccoconcoracnanconoccanonoos 1 1 2 1 EleCErOPhOTFEs S veias 1 12 7 ION a AA Pie 2 2 INSTRUMENT SPECIFICATIONS sissusnic alcista 2 9 8 8 4 POTENTIAL HAZARD ao 5 4 PERSONAL PROTECTIVE EQUIPMENT coc cate 5 5 SPILL AND ACCIDENT PROCEDURES cuidate a 6 6 WASTE DISPOSAL PROCEDURES s ua A 6 Te PROTOCOL ra Aaa 7 TW Sample Ma Brissia A 7 7 1 1 StartUDv sitas oa 7 7 1 2 Set Software Acquisition ParameterS 7 Val A n2 gman 11 7 3 Image ANALYSIS Sxsscsncisdcadnea Sen ane e EA patadas 11 S TROUBLESHOOTING vist RRA 14 APPENDIX VERSADOC USER LOG sion sii 17 VersaDoc Standard Operating Procedure iii DISCLAIMER The materials contained in this document have been compiled from sources believed to be reliable and to represent
6. YPRO Orange SYPRO Red SYPRO Ruby Texas Red Cy2 Cy3 and most flyorescein and rhodamine derivatives Filter 2 530 band pass 60 independent detection of green i e FITC fluorescence in multiple coloured samples PAGE 4 VersaDoc Standard Operating Procedure 5 Filter 3 610 long pass independent detection of red i e Texas Red fluorescence in multiple coloured samples Filter 4 clear filter for white light applications If you are acquiring 1D gel images use the instrument control software Quantity One which includes preset imaging parameters for the following sample types and dyes Nucleic Acid Gels Protein Gels Blotting Densitometry Fluorophores Multiplex and Custom you can define parameters for your own applications To do image analysis and quantitation of your 1D gel you should the ImageLab software not Quantity One ImageLab is available on the VersaDoc computer and additional copies can be obtained from the Instrumentation Technician If you are running 2D gel analysis use the software PD Quest which is capable of both image acquisition and image analysis of 2D gels The software manual and a tutorial of PD Quest cane be found in a folder on the desktop of the computer for the 4000 system Note The computer for the 5000 camera has only has Quantity One installed 3 POTENTIAL HAZARDS The VersaDoc uses a powerful source of UV radiation and may cause damage to unprotected eyes and skin The door saf
7. ation_ 26__Reporting_Procedure pdf s www wlu ca documents 23632 Biosafety_Manual_March_15_2007_Final pdf PAGE 6 VersaDoc Standard Operating Procedure 7 7 PROTOCOL The instructions provided in this protocol are only intended to serve as a reminder all users must be trained before using the VersaDoc for the first time The 4000 camera is always left in the instrument as is used the most often It is highly recommended that you try imaging with this camera first and only switch to the 5000 camera if absolutely necessary Contact the Instrumentation Technician if this is the case 7 1 Sample Imaging 7 1 1 Start Up 1 Turn on the camera 4000 power supply 2 Make sure the VersaDoc enclosure door is closed and then turn on the VersaDoc using the power switch on the right side at the back of the enclosure and allow the initialization to complete wait until only the green power light is illuminated 3 Unless you know that you require the 5000 camera from running previous samples of the same type and the Instrumentation Technician has already changed the camera for you turn on the computer labeled CAMERA 4000 and enter password VD4000 4 After starting up the system it is best to wait 10 or 15 minutes to allow the camera to cool before imaging resulting in better sensitivity Final sample preparation and software acquisition parameters can be set during this time Remember to fill in the log book if you are lea
8. de membranes PVDF The membrane is then immersed in a solution that contains a nucleic acid probe or an antibody The probe or antibody is labeled with a fluorescent dye or linked to an enzyme and will adhere to a specific DNA or RNA sequence or a specific protein antigen on the membrane If using a fluorescent dye the location of the DNA RNA or protein can be visualized by fluorescence excitation and image collection Multiple dyes and antibodies can be used to detect more than one protein at once fluorescence multiplexing If using an enzyme the linked probe or antibody can be detected as a result of chemiluminescence CL or chemifluorescence CF The enzymes usually used are horseradish perioxidase HRP or alkaline phosphatase AP For chemiluminescent detection HRP or AP cause oxidation or dephosphorylation of a substrate on the membrane this then produces light at the location of the nucleic acids or protein of interest If hydrogen peroxide and phenols are present this reaction is enhanced and extended up to 1000 fold and called enhanced chemiluminescence ECL For chemifluorescent CF detection HRP or AP cause oxidation or dephosphorylation of a substrate PAGE 2 VersaDoc Standard Operating Procedure 3 converting it to a fluorescent molecule CF provides a number of benefits including higher signal amplification than direct fluorescence labeling and longer signal lifetime that CL because the substrate only emits light up
9. e make sure the sensitivity sample is hot and fresh Incorrect lens filter used Use the 50 mm high NA lens for best chemi results and use no filter Position 5 or higher Sample on platen with chemi Place sample on chemi tray or tray installed remove tray from sample chamber Adjust f stop to a lower value f 1 4 recommended removed from the lens time Ls o PAGE 15 VersaDoc Standard Operating Procedure 16 REFERENCES Hands on training with Sean Taylor BioRad Field Application Specialist December 1 2010 VersaDoc User Manual http www bio rad com webroot web pdf lsr literature VERSADOC_MANUAL_MP1 REV_F PDF Accessed December 2 2010 Wilfrid Laurier University Environmental Occupational Health and Safety Office 2007 Laboratory Health and Safety Manual www wlu ca documents 23120 Laboratory_Health_ 26_Safety_Manual pdf PAGE 16 VersaDoc Standard Operating Procedure APPENDIX VERSADOC USER LOG PAGE VersaDoc Standard Operating Procedure DATE NAME amp EXTENSION SUPERVISOR SAMPLE DESCRIPTION CAMERA AND LENS TRAY USED F STOP OPTIMIZED PROBLEMS GEL OR BLOT AND USED NUMBER EXPOSURE TIME COMMENTS DYES TAGS USED 4000 amp ZOOM LENS WHITE TRAY 4000 8 FIXED LENS UPPER CL TRAY 5000 LOWER NO TRAY 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000 4000 Et ZOOM LENS 4000 amp FIXED LENS 5000
10. e the red lines and then right click to crop the image b Rotate the image by using the Custom button tool and aligning the crosshairs with the vertical and horizontal planes of the image then right click on the image Return to the Image Tools main menu and click on Lanes and Bands a Under the Lanes Tab i Click on Automatic to allow the software to detect the bands or click on Manual and then enter the number of lanes in the image ii Then use the tools under All Lanes and Single Lane to adjust the width shape and location of the band lanes iii Use Background Subtraction to remove background signal using the software Note a larger disk size of approximately 10 is best for visible stains such as a coomassie gel and a smaller disk size of 2 3 is better for fluorescent and chemiluminescent gels Set the desired disk size and click Apply b Under the Bands Tab i Click on Detect Bands to allow the software to detect the bands or manually add and adjust the bands using the Add Delete and Adjust buttons After automatic detection the bands can still be adjusted or deleted as desired ii To see a more detailed image of the band locations click on Lane Profile at the top of the screen Band locations can be adjusted in this window as well c Return to the Image Tools main menu PAGE 12 VersaDoc Standard Operating Procedure 13 5 If you have a MW standard lane incl
11. es very high resolution The 5000 camera has 512 x 512 pixels less than 0 5 megapixels with a pixel size of 24 mm resulting in lower resolution but higher sensitivity for weak signal applications The 4000 camera is always installed in the Versa Doc contact the Instrumentation Technician if you need to use the 5000 camera Two standard lenses are available for the VersaDoc One is 28 80 mm zoom lens and one is 50 mm f 1 4 fixed lens The zoom lens is suitable for most fluorescence and colorimetric applications and both large and small samples The fixed lens has high light collection efficiency and is recommended for chemilluminescence applications but can also be used for other sample types The fixed lens is always installed in the Versa Doc contact the Instrumentation Technician if you need to use the zoom lens The UV transilluminator drawer should remain in the instrument at all times and is used for fluorescent applications Colourimetric samples require use of the White Light Conversion Screen and chemiluminescent samples should be placed on the upper chemiluminescent tray The sample trays are resistant to most research chemicals but should be washed with a small volume of water and wiped dry with a kimwipe immediately following imaging The VersaDoc 15 equipped with 4 emission filters as follows Filter 1 520 long pass optimized for single colour detection of ethidium bromide DNAStar SYBR Green SYBR Gold Radiant Red S
12. ety interlock and the plastic UV shield protect the user from accidental exposure however it is advisable to use additional protection including UV protective eyewear lab coat and gloves 4 PERSONAL PROTECTIVE EQUIPMENT Standard laboratory protective equipment including latex or nitrile gloves and a lab coat are highly recommended Additional UV protective eyewear is strongly recommended Closed toe and heel footwear constructed of resistant material is also required for all laboratory activities See the WLU Laboratory Health and Safety Manual for additional information on personal protective equipment www wlu ca documents 23120 Laboratory_Health_ 26_Safety_Manual pdf PAGE 5 VersaDoc Standard Operating Procedure 6 5 SPILL AND ACCIDENT PROCEDURES All accidents incidents and near misses involving instruments must be reported as promptly as possible to the Environmental Occupational Health and Safety EOHS Office via the WLU Employee Accident Incident Occupational Disease Report form All incidents that result in critical injuries must be reported immediately to the EOHS Office by telephone ext 2874 Additional details regarding incident reporting can be found in the WLU Accident Incident Procedure The WLU Laboratory Health and Safety Manual provides detailed instructions for dealing with major and minor spills Do not attempt to clean up a spill if you have not been properly trained or if you are unsure of the
13. le chamber application Wrong imaging area selected Verify reselect correct area Bad lamp Replace lamp Dirty optics Ensure that platen filter and lens are clean across the image not working Dust or small particles on the Clean the platen lens and filters spots sample platen or optics White light Image has Chemi Sample tray was not Use the Chemi Sample tray on vertical lines used the UV Filter platen and reacquire the image PAGE 14 VersaDoc Standard Operating Procedure 15 Problem Possible Cause Solution Fluorescent image has Image was taken without being Remove plate from sample high background removed from glass plate Thin polyacrylamide samples can be transferred to exposed x ray film and imaged using UV epi mode 660 NM filter is not in place Install 660 NM filter onto lens Wrong application selected Verify reselect correct application Light leak Check for light leaks ensure Camera Module tightly connected to A fluorescence agarose Use low ul agarose A Ze sample and acquire a control image insufficient 8 o turn housing obstruction obstruction assistance connection Poorly seated lamps Turn the lamps in the sockets to make sure lamps are seated properly in their sockets Epi lamps are not tuming on with age Integration time to Open aperture and reacquire acquire image has the image sample Poor Sample may have degraded Some Chemi samples have a chemiluminescence short lifetim
14. llowing retention of biological VersaDoc Standard Operating Procedure 2 activity Alternatively PAGE can be combined with sodium dodecyl sulfate SDS which denatures and neutralizes the protein allowing separation based exclusively on molecular size Isoelectric focusing combines a pH gradient with applied voltage to separate proteins based on their isoelectic point Two dimensional electrophoresis combines IEF and SDS PAGE to separate proteins based on both isoelectric point first and molecular weight second Following electrophoresis the gel can be stained and visualized or blotted Gel straining techniques indiscriminately stain all of the nucleic acids or proteins in a gel DNA and RNA gels are commonly stained using ethidium bromide and detected using fluorescence Protein gels are stained using coomassie blue or silver for UV detection or Sypro gel stain and CyDyes for fluorescence detection Stained electrophoresis gels only provide information on the size or size to charge ratio or the size and isoelectric point in 2 D separations 1 2 2 Blotting Southern blotting refers to DNA analysis Northern blotting to RNA and Western blotting to proteins this nomenclature is based on the name of the researcher that developed the DNA blotting technique Edward M Southern Blotting a gel after electrophoresis transfers the separated nucleic acids or proteins to a membrane composed of either nitrocellulose or polyvinylidine difluori
15. luminator tray on the bottom Note If possible it is best to remove the glass or plastic plates of a gel sandwich before imaging as the glass and plastic may fluoresce when exposed to UV light and contribute to a higher background signal b If imaging a colorimetric sample it is placed on the White Light Conversion screen which is then placed on the UV tray Note This screen can be removed from the instrument so the sample transfer can also be done in your lab c If imaging a chemiluminescent sample it is placed on the chemiluminescent tray unless it is larger than 8x8 cm in which case it should be placed on the UV tray Note This screen can be removed from the instrument so the sample transfer can also be done in your lab If NOT imaging a sample on the upper chemiluminescence tray make sure to remove it Set the camera iris by adjusting the aperture ring aka f stop If imaging samples with weak signals i e chemilumnescent samples set the f stop to a lower number this opens the iris to allow more signal to reach the detector If imaging samples with a strong signal i e colorimetric samples set the f stop to a high value to avoid saturating the detector Refer to Table 7 1 for recommended settings PAGE 8 VersaDoc Standard Operating Procedure 9 Table 7 1 Recommended VersaDoc 4000 Settings for Common Sample Types Recommended Settings Signal Type and Exposure np ras Expected Strength
16. mage Report to view the collection parameters for that image Note The brightness and contrast of each image is automatically adjusted after image collection but this affects the appearance of the image only not the actual density 14 To check the density of the last image right click on the image and select Density at Cursor then left click on one of the darkest bands in your image The maximum density is 65 000 and an optimized image will have a maximum density close to but not at 65 000 a If the maximum density in the last image with the longest exposure time is far below 65 000 re collect the image with a longer exposure time or a smaller f stop setting or if doing a chemilumnescent sample change the selected method to chemi Super or chemi Ultra If this does not result in sufficient sensitivity you need to switch to the 5000 camera Contact the Instrumentation Technician If the maximum density in the image is 65 000 the camera detector was likely saturated and the pixels appear red Select PAGE 10 VersaDoc Standard Operating Procedure 11 an image with a shorter exposure time or re collect the data with a larger f stop setting 15 Choose the optimized exposure time and click on Acquire 16 When a satisfactory image has been obtained save the final image as a TIFF file and a 1sc file Image analysis can be conducted in ImageLab see section 7 3 7 2 Cleanup The sample trays are resis
17. ng an arrow click on the Annotation Tools button 9 When all quantitation is complete click on Analysis Table or Report to generate a list of the final results 10 Save the quantified image under a different name from the original file PAGE 13 VersaDoc Standard Operating Procedure 14 8 TROUBLESHOOTING Table 8 1 lists some of the problems commonly encountered when using the VersaDoc For additional troubleshooting tips please see the VersaDoc manual Table 8 1 Common sectioning problems and causes VersaDoc User Manual Possible Cause Solution VersaDoc 15 not Power is not supplied or the Ensure power is supplied to the responding to host system is not switched on system and that the switch is computer turned on Serial USB or AIA cable is not Reconnect Serial USB AIA cables connected properl and ensure it is seated proper Camera power cable is not Makes sure that the cables are connected proper connected and seated properl Change PCI slot to which the digitizing card is connected NA AREA AR Turn off all components and restart in opposite sequence program or initiation init file Image is not visible on The Transform function in the Set to a lower maximum value the monitor or only low software is set too high signal counts are detected Lens cap 15 covering lens Remove lens cap Insufficient integration time Integrate sample for a longer time Chemi tray not removed when imaging nonchemi samp
18. not adjust the focus ring on the camera to match up the other red arrow sticker 11 Under Step set the exposure time Please Note The Iris f stop and exposure time have similar influences on your images For example a brighter image can be acquired by a smaller f stop numer or longer exposure time You can adjust both parameters to get best images a Refer to table 7 1 for recommended exposure times and click Preview to see the image PAGE 9 VersaDoc Standard Operating Procedure 10 b For a new sample type select Optimize exposure and enter the following Optimize cannot be used for multi channel imaging make sure Highlight saturated pixels is selected Save Images is not selected i For a chemluminescent sample i e Western blot enter 100 for the starting exposure 600 for the total exposure and 10 for the number of exposures ii For a colourimetric sample i e coomassie blue or silver enter 0 1 for the starting exposure 10 for the total exposure and 20 for the number of exposures iii For a fluorescent sample i e sypro ruby GFP or a UV sample i e ethidium bromide enter 1 for the starting exposure 10 for the total exposure and 10 for the number of exposures 12 Press OK The images taken with different exposure times will be visible on the screen as they are collected 13 Click Window gt Tile to see all of the collected images You can right click on an image and select I
19. on fluorescence excitation A CF substrate can therefore be imaged several times See Figure 1 1 for a flowchart of basic blotting and detection methods See http www molecularstation com protein troubleshooting western blots for useful troubleshooting tips for Western Blots Electrophoresis Agrose Gels Native PAGE SDS PAGE IEF Strand Mass to Molecular Isoelectric length charge ratio size point ES ki b E UV Blue Silver Stain light dyes Ethidium Stain and Stain and _ Coomassie Blue Bromide visualize visualize Sypro Rub Blot and Identify mm m 7 CyDye DiGE Reporter Enzymes Fluorescent Dyes Enhanced Colorimetric chemiluminescence detection detection Figure 1 1 Separation of DNA RNA or protein samples using electrophoresis is followed by blotting and identification of specific fragments using reporter enzymes or fluorescent dyes PAGE 3 VersaDoc Standard Operating Procedure 4 2 INSTRUMENT SPECIFICATIONS The VersaDoc is a quantitative imaging system for digital image acquisition from single and multi colour fluorescent chemifluorescent and colourimetric blots gels or plates up to 25 x 25 cm in size The system includes a light tight enclosure and uses one of two cooled CCD cameras designed for high sensitivity and high resolution The 4000 camera has 2 184 x 1 472 pixels 3 2 megapixels and a pixel size of 6 mm which provid
20. ration for the VersaDoc Imaging System henceforth referred to as the VersaDoc 1 2 Theoretical Background The VersaDoc can be used to image DNA RNA or proteins on electrophoresis gels blots or plates Electrophoresis can be conducted in a variety of ways to obtain different information including agrose gel electrophoresis native polyacrylamide gel electrophoresis PAGE sodium dodecyl sulfate PAGE SDS PAGE isoelectric focusing difference in gel electrophoresis DIGE and 2 dimensional electrophoresis Following separation by electrophoresis DNA RNA or proteins can be stained and detected or further transferred onto a blot to allow for more specific detection Blotting techniques include Southern Northern and Western blotting and a wide variety of visual and fluorescent detection methods 1 2 1 Electrophoresis Electrophoresis of nucleic acids is typically carried out on agrose gels and separation is based entirely on molecular size Polyacrylamide gels are used for nucleic acid separation for short DNA or RNA strands and provide high resolution for applications such as DNA sequencing Protein electrophoresis encompasses a much wider variety of techniques Proteins can have a variable charge depending on the chemical composition and the environmental conditions Native PAGE separates protein fragments or peptides based on the mass to charge ratio of each fragment and does not alter the secondary structure of the protein a
21. tant to most research chemicals but should be washed with a small volume of water and wiped dry with a kimwipe or soft sponge IMMEDIATELY following imaging 7 3 Image Analysis Image analysis can be carried out in ImageLab This software is available on the VersaDoc computers and a copy can be optained from the Instrumentation Technician For informative tutorials on ImageLab see Image Analysis https sas elluminate com site external jwsdetect playback jnlp psid 2010 09 07 2117 M 9EF814CC7FF 199 166934248F 1 98E09 vcr amp sid voffice OR Select Tutorials when you open the ImageLab software The following protocol provides a basic outline for densitometry and MW analysis 1 Click on the ImageLab software and open the saved image a If the sample was labeled with multiple dyes or you want to merge more than one image of the same sample then open all of the images for that sample and select Image Tools then Merge Note Merged images are best used for illustration purposes ONLY All quantitation should be done on original un merged images PAGE 11 VersaDoc Standard Operating Procedure 12 2 If analyzing a blot or gel follow steps 3 through 6 If analyzing a plate skip to step 7 All image analysis options are listed in the toolbar on the left side of the screen It is usually best to start at the top of the list with Image Tools a Crop the image as desired using the Crop button mov
22. the best opinions on the subject This document is intended to serve only as a starting point for good practices and does not purport to specify minimal legal standards No warranty guarantee or representation is made by Laurier as to the accuracy or sufficiency of information contained herein and Laurier assumes no responsibility in connection therewith VersaDoc Standard Operating Procedure iv ACKNOWLEDGEMENTS The following individuals of Laurier contributed to the writing editing and production of this manual Gena Braun Instrumentation Technician Jiangxiao Sun Instrumentation Technician This manual was prepared for Laurier Any corrections additions or comments should be brought to the attention of the Instrumentation Technician at 519 884 0710 ext 2361 Issued May 2011 Revised April 2012 VersaDoc Standard Operating Procedure 1 1 INTRODUCTION 1 1 Purpose of the Standard Operating Procedure This standard operating procedure SOP is NOT a substitute for training and or reading the appropriate manuals before use All principle investigators and supervisors should document that training has been received by students and staff who will be using the VersaDoc Imaging System This SOP 15 intended to promote consistent and safe use of the VersaDoc Imaging System This SOP covers the potential hazards personal protection requirements spill and accident procedures waste disposal considerations and instrument ope
23. uded in your image click on MW Analysis If not skip to step 6 for band intensity quantitation densitometry a e Use the checkboxes under the lanes to indicate which lane includes the MW standards Click on the Change button to select the appropriate set of MW standards If the desired standard set is not listed add it using the New button The resulting curve can be viewed using the Standard Curve option from the menu at the top of the screen To see the calculated MW values click on the desired lane and then choose Analysis Table from the top menu Alternatively click on Report to generate a complete list of all lanes along with images Return to the Image Tools main menu 6 To perform densitometry click on Quantity Tools a Use the Relative tab to determine the density of each band relative to one of the bands in the image Use Select to choose the reference band Use the Absolute tab if the actual concentrations of more than one band are known Use Select to choose the known reference bands and enter the known values At least two bands must be chosen to create the calibration curve The resulting curve can be viewed using the Standard Curve option from the menu at the top of the screen Return to the Image Tools main menu 7 If analyzing the image of a plate use the Volume Tools button 8 To add any text or highlight a band usi
24. ving for sample preparation indicating to others that the instrument is in use 7 1 2 Set Software Acquisition Parameters 1 Start the Quantity One software from the desktop 2 Select File gt VersaDoc 3 Under Step I click on Select and choose the desired application a If you are analyzing a colorimetric sample you will be prompted to insert the White Light Conversion Screen on the UV tray The Conversion Screen is stored in the cupboard below the imager and should be placed carefully on the UV tray with the rubber gasket side down PAGE 7 VersaDoc Standard Operating Procedure 8 4 7 b If you are imaging a chemiluminescent sample select chemi high initially and if the image collected in steps 13 and 14 15 still very weak try chemi super or chemi ultra If you are analyzing a sample with multiple dyes set up a channel for each dye by clicking on each channel tab selecting enable channel and choosing the desired application for that channel If none of the applications satisfy your sample you can select Custom then Edit to create a new VersaDoc application Fill in the required information Name Filter Illumination Gain and binning click on OK Check that the sample trays are clean and place the sample onto the correct tray Note if the trays appear dirty wipe with a kimwipe and DI water a If imaging a fluorescent sample place it directly on the UV transil
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