What i referred in literature was temperature decreases with successive cycles in 1st phase followed by 2nd phase where cycles continue with final temperature achieved in 1st phase. I am still getting high non specific bands. Some are saying we keep annealing higher than tm in 1st phase and in 2nd phase we decrease. Which protocol is authenticate and correct ?
Hi,
Your reference sounds is good. If you getting non specific bands, try doing gradient PCR first to find out optimum annealing temperature. Also double check your GC content if amplicon GC ~60 and more consider using DMSO in reaction. Alternatively, you can also think of nesting PCR.
you reference is good. If you are still facing with non specific band, increase the temperature from which you start. ie try to start at 65°C, then decrease up to 55 and do 20 additional cycles at 55. Then if you have no bands at all, try a nested PCR with two internal primers.
(PS: what is the Tm of your primers?)
Hi,
Touch Down PCR is a modification of PCR in which the initial annealing temperature is higher than the optimal Tm of the primers and is gradually reduced over subsequent cycles until the Tm temperature or “touchdown temperature” is reached. Increases the specificity of the PCR reaction.
Additional details:
https://youtu.be/5YWKdMngzkk
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