Hi All,
When I optimize PCR protocols, I find the peaks of main products are getting shorter and shorter while the peaks of non-specific products are getting larger and larger. Does someone know why that happens? Thank you so much!
Hot-start PCR also reduces non-specific binding and amplification.
Dear Nan,
What is the Tm of your primers?
What is the annealing temperature do you use?
Dear Nan,
Yes, you could amplify a longer segment by accounting for flanking regions on either sides of your GOI. By this way you will have got rid of the non specific binding region. The second set of primers will then amplify only your GOI, and since you do not have the non specific region in the second run, you will only have your GOI amplified!
P.S. Have you tried increasing specificity in the existing PCR programme?
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