I have a trio of PCR primers, one forward and two reverse with few bases of separation, without overlapping, it only works good when I use all primers, and sometimes when I use a pair, PCR was perform with a polymerase from New England and also with a Qiagen top taq master mix. Ideal annealing temperature was determinated by gradient , PCR product was sequencend and is correct.
If I understood correctly, the reverse primers are partly overlapping each other? If so, this may suggest a mistake in primer sequence (wrong annotation, mistake in order, or in synthesis).
if we considered F1 & R1 and R2 I only get the bigger size PCR product between F1 and R2, intermediate R1 primer is like a helping to reaction of others. I only get one PCR product, one band with size between F1 and R2
Did you check whether any primer dimers are being built? This seems a plausible explanation, too. OR self-dimerization of any pf the primers.
http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
Are you adding all three primers in the initial mix or are you adding R1 sequentially?
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