Why is my smaller band too faint? Genomic PCR not working! - Just trying to genotype some mice... (Apr/01/2006 )
Hi,
I'm at my wit's end and would appreciate any and all advice. I'm trying to get a mouse genotyping PCR protocol working. The mice and the protocol come from an extremely well respected genetics lab - but I can't get the protocol to work! I'm not a molecular biologist, but I've been trying to learn as much as I can to make this work.
Here's a summary of what I do:
DNA extraction using a Qiagen kit - others have used and love this kit; the lab that developed the mice does prot K digestions & phenol/chloroform exrtractions without a kit.
Autoclave tubes for DNA, PCR, PCR primer aliquots.
PCR on ~300-900 ng mouse genomic DNA.
Use same PCR reagents from same vendors as described by the lab the protocol was developed in: regular Taq from Promega, Invitrogen Buffer E (has a specific Mg2+ conc. & pH; which says to me that they had at least some kind of problem, too!), DMSO, no extra Mg2+, etc.
Total rxn volume: 25 ul.
Set up rxns on ice, add Taq at last minute, & start PCR.
Thermocycling conditions: 1 cycle: 2 min @ 94C; 35-38 cycles: 30 sec @ 94C, 30 sec @ 60C, 1 min @ 72C; then 4C.
Run PCR products on a 1.5% agarose gel, made & run using LB Buffer (Faster Better Media), made fresh each time. I usually add 25-50 ug EtBr to the gel.
(And... I always wear gloves, & I'm a careful & experienced pipetter.)
I use 3 primers: 1 specific to WT mice, 1 specific to the KO mice, and 1 common primer. The common primer is at twice the concentration of the other 2 primers (0.3 ul, 100 uM).
What I'm looking for: KO band: 600 bp. WT band: 400 bp. I'm mating heterozygous mice that have normal proportions of each genotype as offspring. So, I should see a KO band in ~75% of mice, & a WT band in ~75% of mice.
Instead, I see a strong-to-blazing KO (upper) band in ~75% of my mice, and a weak-to-barely-there WT (lower) band in at most 1/8 to 1/4 of my mice. I got better reactions *once*, and identified a few clear hets and WT mice - although the WT band was still a bit too faint for comfort in some mice. But, I've never been able to get my reactions to go even that well since, using the same & new DNA.
At this point, I have tried just about everything I know to do. I have tried varying the annealing temp from 47-60C - got some non-specific bands at lower temps, but surprisingly, not a brighter WT band. I have tried increasing the WT primer concentration to 2x & 10x - that just made my KO band fainter. I've tried new tubes of everything more than once. I've tried the protocol in a Stratagene PCR machine with a robotic arm and in a regular Perkin-Elmer machine - no difference. I've tried using more DNA - that helps, until the bands disappear and are replaced with a smear & perhaps a mega-kb band when I get up to ~1.2 ug DNA or more. I've tried lengthening the times at 94C, 60C & 72C by 30 sec - no difference. I've tried adding more EtBr & taking longer pictures - that helps to a limited extent, but really, the band's just not there; I just get more background.
What am I doing wrong? What can I do to make this work??? Any help will be appreciated!
These are just guesses, but some things to check:
* I was unsure about what you meant when you said you autoclaved tubes for your primer and DNA. Did you mean you autoclaved them prior to filling them, or that you autoclave your primers? In general, I don't do anything with my tubes -- they come clean, and if you really need them sterile (rare) then you can buy them sterile. Autoclaves cause cross contamination.
* Your bands may be disappearing due to migration of EtBr out of your gel. You can test this by post staining your gel in buffer + EtBr, or by adding EtBr to the positive buffer well in your gel box. EtBr migrates toward the negative electrode (opposite direction from DNA) and can destain bands of short length.
* You don't say which Qiagen kit you are using. I presume it's for preping genomic DNA, not plasmids.
I'd go with prot K and phenol/chloroform, myself.
Thanks so much for your reply.
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* I was unsure about what you meant when you said you autoclaved tubes for your primer and DNA. Did you mean you autoclaved them prior to filling them, or that you autoclave your primers? In general, I don't do anything with my tubes -- they come clean, and if you really need them sterile (rare) then you can buy them sterile. Autoclaves cause cross contamination.
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I have autoclaved my tubes before filling them. From the discussions I've had with various people about autoclaving tubes, opinions seem to vary so greatly. You are the first to mention the issue of potential cross contamination. Others have said definitely to autoclave tubes (eliminate potential organisms), and others have said definitely not to autoclave tubes (could warp tubes). Clearly, it can't be that bad not to, since so many people, yourself included, swear by not doing it!
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* Your bands may be disappearing due to migration of EtBr out of your gel. You can test this by post staining your gel in buffer + EtBr, or by adding EtBr to the positive buffer well in your gel box. EtBr migrates toward the negative electrode (opposite direction from DNA) and can destain bands of short length.
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I suspect this might be a very relevant point - thank you!
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* You don't say which Qiagen kit you are using. I presume it's for preping genomic DNA, not plasmids.
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Yes, it's for preparing genomic DNA from animal tissues.
Thank you so much again for your help, and for taking the time to write. I really appreciate it.