I'm getting very less amount of amplification in overlap extension PCR. Fragments I'm using as template are of 516bp and 3.2kb. Please suggest the ratio of mixing of these two fragments. And also about the annealing temperature (i'm using 55 'C).
Ratio i'm using= 20ng:120ng (516bp and 3.2kb)
95 'C- 5mins, 95'C- 30s, 55'C- 30s, 72'C-1 min 30s, 72'C 5 mins
(5 cycles without primers, 30 cycles with primers)
Q5 Polymerase
Check the Tm of overlapping region between these two fragments. Use that Tm to simply extend the mixed fragments for 4-5 PCR cycles. After these 4-5 cycles add the primers designed for full-lenth amplification and perform the PCR for 30-35 cycles. We perform such extension amplification by this way only.
I usually include the outer primers right from cycle 1 and it generally works well.
I would also recommend trying to decrease the template DNA amount. Make sure the primer molar concentration is >10x higher than the template concentrations. This is to make sure that the primers can outcompete the opposite strand of the template during annealing.
I'll also point out that Q5 polymerase does not have 5'-3' exonuclease activity or strand displacement activity. This could mean that primer extension could be partially blocked at each cycle by overlapped template stands. If you don't need super high fidelity amplification (ie. you'll be picking and sequencing several clones for correct product), I'd recommend using basic Taq polymerase (which does have this stand displacement activity).
Good luck
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