PCR Genotyping Problem - (Sep/09/2008 )
I recently received a knockout mouse from an another lab. They provided 3 primers that distinguish between WT and KO alleles. The only information they provided us was the sequence of each primer and the annealing temp... so we assumed it was a pretty straight forward PCR... of course, not the case.
We were unable to see either WT ot KO bands in tail DNA template, so i figured it was an issue with the multiplex PCR. When I separated the primers I still was unable to see the band. So we thought it was template and tried the PCR with DNA from the liver of a sacrificed mouse and it worked like gold. The only problem is, we need the mouse to be alive!!!
We have adjusted Mg ion concentration, added DMSO, used different PCR kits, prepared the DNA differently (pure and crude tail lysates). I have also tried toe and ear punch DNA and it did not work either. Since the DNA from liver works so well, I am inclined to believe its a DNA prep issue as opposed to PCR conditions... but I am at a loss!
Getting this mouse was not the easiest to do and we really do not want to have to go to their lab for details!!!
Does anyone have any suggestions or recommendations on how I can come up with a standard protocol to PCR genotype this mouse?
The first thing I would try is 100x and 1000x dilutions of your template DNA, since the PCR will still amplify it, but the PCR inhibitors will be diluted to insignificance.
I would also check out those primers. Blast the sequences and see what you get.
Are you quantifying the DNA after purification? I genotype bits of tail all the time with no problems. You should end up with more than enough DNA for PCR doing a basic extraction, but to check, put 1µl in a nanodrop to see if you have DNA or not. (I digest bits of tail overnight in a proteinaseK solution, then spin down the cellular debris, and purify DNA from the supernatant using isopropanol precipitation, followed by a desalt with 70% ethanol. Resuspend in 50 µl H2O or 10mM Tris-Cl pH 7.5.)
I was going to suggest this, too. The fact that your PCR worked on liver DNA suggests they might be okay, but I ALWAYS BLAST new primers I'm given for genotyping to see what I'm getting myself into.
Two anecdotes from my own experience:
1) We received a genotyping protocol for a new mouse line and had absolutely no luck getting any PCR product. Turns out they'd given us the sense strand sequence for ALL primers (only found that out from doing a BLAST). This experience is what got me into the habit of always BLASTing sets of primers I didn't design myself.
2) I was once having genotyping PCR troubles, and discovered on BLASTing that the sequence was very GC-rich. Modified the PCR protocol to include betaine, and it's worked ever since.
Appears to be a template specific problem. I assume you have tried gradient PCR with various annealing temperatures? When I used to do PCR with difficult DNA samples (some old blood preps) it helped if I heated the DNA sample to 50 degrees for about 10 minutes before adding an aliquot to the PCR reaction. Recently I also found out that Bioline Taq polymerases are very efficient with difficult PCRs (check their web site, they have free samples). As has been suggested already, dilute your sample a lot before doing a PCR (a few ng is enough!).
Anyway, I would still suggest you to order new oligos. I have learned that when a PCR does not work in the first couple of tries (I usually do a gradient in the beginning), then it would be a waste of time to optimize it any further. Usually new oligos save you a lot of time.
Thanks for the great replies everyone... I am running a PCR at the moment with dilutions of the template as suggested by Phage. I really think that there is some kind of contaminant in the tail PCRs that is not present in the liver.
As for the other comments, I do not believe it to be a problem with primers because it works perfect with liver genomic DNA template, so the primer sequence, annealing temp, and general PCR conditions seem to be ok. Also, there is DNA present in the sample because it does amplify other primer pairs, just not the pairs I need it to!
I will keep you updated on my findings after I run the gel today... hopefully this thread will be closed soon there after
No dice... a very very very very faint band in the 1:100 dilution (1:500 and 1:1000 did nothing).
I FIGURED IT OUT!!!!
I dont know why exactly, but HOT START Taq from TaKaRa worked beautifully with tail and ear punch DNA! I was not using hot start enzyme before...
Thanks for the suggestions on this thread!