I am using nested PCR to determine the alleles present in my parasite population for a specific gene. This has gone well, but one gene has given me an issue.
The initial PCR for gene MSP1 resulted in a product of 477bp which was expected. The following reaction should produce products between 100-300bp, however, I am seeing those amplicons in some samples as expected but there some samples have a band around 500bp.
The primers are from literature and I have not designed them.
Is it likely the primers are non-specific? In this case where the expected amplicon size for the allele is known do I then ignore these larger amplicons?
Any help would be appreciated
Edited to add: I'm using the PCR conditions specified for the mastermix with the annealing temperature as determined by the supplier's annealing temperature calculator available online.
did you gel purify the first PCR product? If you are just diluting the reaction you could have WAY too much of the primary primers in the reaction and they are continuing to amplify in the nested reaction.
Julie Ann Dougherty I did not, that makes sense. I'll give it a try and hopefully it helps the problem
I agree with Julie Ann Dougherty . I normally diluted first round product 1:100 then used 1ul and 20-25 cycles second round pcr. Too many cycles can result in a high molecular weight smear with overamplification
- Badges
- Science method