Appendix B: Troubleshooting the DIG System
Contents
1. C 44 84 164 244 Possible causes Air bubble was trapped between the membrane and gel during blot transfer Depurination with HCl not performed if missing bands are gt 5 kb Recommendation Always set up the blot transfer sand wich carefully and eliminate all air bubbles trapped between the surface of the gel and membrane If DNA on gel is large gt 5 kb treat the gel with 0 25 M HCl 10 20 min before starting blot transfer This depurinates the DNA so it will be fragmented dur ing the subsequent alkaline denatura tion step and will transfer easily to the blot see Section 3 1 2 2 page 91 of Chapter 2 for details Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations Problem 12 Unwanted rRNA Hybridiza tion Signals on a Northern Blot Data courtesy of M Block University of Hamburg Germany 28s 18s Possible cause Labeled probe contained unwanted RNA or chromosomal DNA sequences from E coli which bound ribosomal RNA in the samples Recommendation Prepare labeled probe from a plasmid DNA preparation that is free of contam inating chromosomal DNA and RNA e g purify the plasmid with the High Pure Plasmid Isolation Kit Instead of a DNA probe use an RNA probe prepared by transcriptional label ing as in Section 2 3 page 68 of Chap ter 2 Do not exceed the recommended target RNA loads For DNA probes 5 ug total2. DIG System 1 Possible Problems and Recommendations Problem 3 Irregular Cloudy Background Possible cause Uneven distribution of probe during hy bridization caused by not using enough hybridization solution or by letting the membrane dry during incubation Recommendation Do not add probe directly to prehybrid ization solution Do not allow membrane to dry between prehybridization and hybridization For instance do not pour off the prehybrid ization solution until the hybridization solution is ready for immediate addition to the membrane Use at least 3 5 ml of hybridization solu tion per 100 cm of membrane Note If using roller bottles for incuba tion use at least 6 ml hybridization so lution per bottle Shake the hybridization container dur ing the hybridization incubation Caution Make sure that the hybridi zation bag lies flat in the bottom of the water bath 185 N t oT D zi 2 lt x N Q x e lt x Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations 186 Problem 4 Irregular Smeared Grainy Background Possible causes Non uniform distribution of chemilu minescent substrate e g due to per forming incubation while membrane wrapped in plastic wrap Wrinkles in the hybridization bag caus ing uneven contact between membrane and X ray film Drying of membrane during chemi luminescent visuali
3. RNA or 500 ng mRNA For DIG labeled RNA probes lug total RNA or 100 ng mRNA 19 N t oT D zi 2 lt x N ct iS x 2 lt x Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations 192 Problem 13 The Membrane Strikes Back Possible causes Lack of care in handling membrane dur ing procedure Note Every scratch touch or gouge on the surface of the membrane will be made visible during the chemilumines cent detection visualization procedure Blot that had previously been hybrid ized only with radioactively labeled probes was rehybridized with a DIG la beled probe Note All these background marks were not visible in the radioactive detec tion procedure Paper towel print Finger print Specific signal A Forceps marks Recommendation Be very careful when handling the blot during a DIG procedure Handle it only by the edges and only with gloves and forceps Do not touch the experimental portion of the blot with anything Always start with a fresh blot when per forming a DIG procedure for the first time do not reuse a blot from a previous radioactive detection procedure Mem brane damage that is invisible during a radioactive procedure may be visible in a DIG procedure
4. Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations Problem 1 Too weak or too strong signals A B Possible cause Chemiluminescent assay exposure time too short A or too long B Recommendation Increase for A or decrease for B the amount of time you expose the blot to X ray film or in the Lumi Imager Note The display range tool of the Lumi Imager allows adjustment of the signal in tensities see Section 3 1 2 e t i x 2 2 lt x 184 Problem 2 Uniform High Background Possible causes Probe concentration was too high dur ing hybridization most likely cause Template DNA was contaminated lead ing to impure probe Recommendation Reduce probe concentration Perform a mock hybridization with dif ferent concentrations of probe as de scribed in Part 2 of Appendix B page 194 to determine the amount of probe that gives the most signal with the least background Never use the entire yield from a label ing reaction to analyze a single blot as you might for radioactive analysis Purify the probe with proteinase K treatment and or filtration through a 0 45 um cellulose acetate filter or use the High Pure PCR Product Purification Kit e Purify the template by phenol extrac tion and ethanol precipitation Appendix B Troubleshooting the
5. Membranes from Roche Molecular Biochemicals N Q z z D a T Recommendation Wash the membrane briefly in 2x SSC before baking Do not bake the membrane Instead fix the DNA by UV crosslinking then rinse the membrane with water as de scribed in Section 3 1 2 2 page 91 of Chapter 2 188 Problem 7 Gray Circles above Bands Data courtesy of Dr Bacchetti and Dr Marusic McMaster University Canada Possible cause Membrane was too dry before chemilu minescent substrate was added The sub strate dried at several spots on the mem brane leading to the gray circles Recommendation Do not let the membrane dry even slightly before adding the chemilumi nescent substrate Cover the membrane with the second sheet of the folder or bag immediately after you add the chemiluminescent substrate as described in Section 4 1 page 112 of Chapter 2 Even a little dried substrate can lead to gray circles especially if you are using CDP Star Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations Problem 8 Spots on the X ray Film in Areas Not Covered by Membrane Possible cause Electrostatic charge on the outside of the sealed hybridization bag Recommendation Wipe the surface of the sealed bag with 70 ethanol before incubating it with the X ray film When handling the membrane always wear gloves Use forceps never fingers to grip the me
6. ations Problem 5 cont Problem 6 Spotty Background Possible cause 2 Possible cause 1 Data courtesy of Dr Bacchetti and Dr Antibody contained a precipitate when it Marusic McMaster University Canada was applied to the membrane Membrane dried while in blocking solu tion and stuck to side of incubation tray Recommendation Never let membrane dry at any stage of the prehybridization hybridization or Recommendation detection procedures Before each use centrifuge the antibody Always use enough liquid in each incu preparation in its original vial for at bation to cover membrane completely least 5 minutes at 10 000 rpm Take an Control the membrane occasionally aliquot from the surface of the superna during incubations especially those tant for the antibody dilution with agitation to ensure it doesnot dry Be sure the Detection Buffer used after or stick to the incubation tray the antibody incubation step does not contain Mg ions N Q oT D zi 2 x 187 Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations Problem 6 cont Problem 6 cont Possible cause 2 Possible cause 3 Membrane was unsuitable for nonradio Data courtesy of T Ruckes Institute of active assay Virology Erlangen Germany Salt crystals from 20x SSC were baked into membrane before the detection pro cedure Recommendation Use positively charged Nylon
7. mbrane Grip only the edges of the membrane never the center even with the forceps 189 N t oT D zi 2 lt x e Q i x a 2 lt x Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommendations 190 Problem 9 No Background on Membrane but Strong Nonspecific Smear across Lanes Data courtesy of Dr B Hengerer Ciba Geigy Switzerland Possible causes Probe binds to some other nucleic acid in sample besides the desired target Too much target nucleic acid on gel Target nucleic acid partially degraded Recommendation Reduce the amount of target loaded on gel Reisolate target nucleic acid and check the sample for degradation before using it in the hybridization experiment Problem 10 Diffuse Bands Ps i f g n ray gt pe a Possible causes Incomplete transfer of nucleic acid dur ing blot transfer Mashing of gel during blot transfer Partial drying of gel during vacuum transfer Recommendation During a capillary transfer make sure the weight is evenly distributed over the surface of the blot and does not mash or otherwise distort the gel e During a vacuum transfer be sure the vacuum is constant Also be sure the vacuum is strong enough to effect the transfer before the gel dries unevenly Problem 11 Missing Bands Data courtesy of Dr B Hengerer Ciba Geigy Switzerland
8. zation procedure Note The grainy appearance of the background indicates that the drying occurred during the chemiluminescent procedure rather than during hybrid ization Drying during hybridization leads to a cloudy background as shown in Problem 3 Recommendation Do not wrap membrane in plastic wrap during incubation with chemilumines cent substrate Spread the chemiluminescent substrate uniformly over the surface of the mem brane as described in Section 4 1 page 112 of Chapter 2 Before exposing bag to X ray film flat ten any wrinkles between blot and membrane by rolling a pipette over the surface of the bag Carefully seal the damp membrane in a folder or bag during the incubation ex posure to X ray film Check the seals to make sure liquid cannot leak Problem 5 High Background on Only Part of Membrane Possible cause 1 Data courtesy of Dr Bacchetti and Dr Marusic McMaster University Canada Drying of membrane during chemilumi nescent detection procedure Recommendation Do not wrap membrane in plastic wrap during incubation with chemilumines cent substrate Plastic wrap cannot be sealed and will allow membrane to dry out Carefully seal the damp membrane in a development folder or hybridization bag during the incubation exposure to X ray film Check the seals to make sure liquid cannot leak Appendix B Troubleshooting the DIG System 1 Possible Problems and Recommend
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