Is there a different type of primer i have to use when running PCR with genomic DNA compared to cDNA?
February 1st, 2009 | by DistanceRunner |bob oso asked:
Im still in school but when i run a PCR, I normally isolate RNA first then RT to cDNA and run PCR with that. This time i isolated the genomic DNA from the sample and ran PCR using the beta-actin primer i always used. Dimers were faintly visible on my gel but nothing really came up like it normally would have or should have. I was told that when running PCR with genomic DNA, a different kind of primer had to be used; one that is specific to genomic DNA. Im confused as to why it would matter or if it matters at all.
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Im still in school but when i run a PCR, I normally isolate RNA first then RT to cDNA and run PCR with that. This time i isolated the genomic DNA from the sample and ran PCR using the beta-actin primer i always used. Dimers were faintly visible on my gel but nothing really came up like it normally would have or should have. I was told that when running PCR with genomic DNA, a different kind of primer had to be used; one that is specific to genomic DNA. Im confused as to why it would matter or if it matters at all.
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2 Responses to “Is there a different type of primer i have to use when running PCR with genomic DNA compared to cDNA?”
By pedestrian on Feb 3, 2009 | Reply
Wow. I”m a lil confused here.
By Lisa on Feb 3, 2009 | Reply
It depends on your definition of primer types, and how you originally designed your primers. If the primers you use on your cDNA were designed to cover exon/intron boundaries, then those primers are not going to work right on genomic DNA because the introns are present. In that case you would need to design new primers. These will be very similar in that they’ll be 20-ish-mers of DNA, but will have a different sequence.
Since all you’re seeing is primer-dimers, I’m guessing this is the case. You could try messing around with the annealing temperature and MgCl2 concentration though if you think it should be working, and compare the sequences of the primers, cDNA, and genome to make sure they’re all lining up and matching.