Working to genotype CRISPR-Cas9 triple mutants. One of the three genes amplifies very well, the other two won't. I have tried three different primer pairs for each of the two genes that won't amplify well, and only specific samples will work, while almost all are working for the third gene. I have tried large amplicon (~500bp) and smaller (~200bp) primers. It seems like selective samples will amplify, any ideas what would be causing PCR to be inhibited in most of the samples for two fo three genes? (note, the two genes sit right next to each other in the genome, but I have tried to design primers with limited 3' matches in NCBI blast). I don't think it is the DNA preps since they work with the one primer, but then what would be causing PCR to only sometimes work for the other two genes (less than 10% of the time)?
One reason for primers not amplifying is that the 3' end of the primer cannot anneal. Perhaps the few samples that amplify have the correct sequence and the rest have a polymorphic mismatch stopping the pcr from working. It may be that the sequence of the region between the 2 genes is a bad area for primers. Can you move the primers out to amplify both genes in one amplimer when sequencing may provide the answer to non amplification of the individual genes?
If the primer mismatch is small then lowering the annealing temperature may allow the primers to anneal. Try a pcr at 6-10c lower than your current annealing temperature
Paul Rutland explained really well.
The few samples that worked, did you sequence them? Were they mutants or WT?
I guess the target of the for the sgRNA is sitting in between the two primers (at least 30-50 bp from the primer sequences)?
Thank you for the advice! So what I think that I found is that CRISPR was making two DBS breaks within 6kb (that's how close these two genes are), and that entire piece of DNA was being excised in the repair mechanism. I amplified samples with a reverse primer from one gene and the forward from the other gene, and got a 500 amplicon band where it would be 6kb in a WT, so I think I found the answer to my PCR troubles!
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