My protocol
Primary PCR (NP6/21): Initial Denaturation at 94C, 2 min.; 30 cycles Denaturation 94C 30 sec, Annealing 65C 1 min., Extension 72C 1 min.; Final Extension 72C 5 min.;
Nested PCR (NP7/10): Identical to Primary PCR. We used 1 ul of primary reaction in the nested.
When you order primer oligmers, check candicate primers first before you order them. There are online tools which can be used to check *primer dimers*. For example, this one from Sigma-Aldrich (see attachment), and its link below:
https://www.sigmaaldrich.com/technical-documents/articles/biology/oligo-evaluator.html?gclid=CjwKCAiAwrf-BRA9EiwAUWwKXl7pEV66NQSXRLrOtX7bCylkiLnP4hbaDDyFLvfSBDAt9YOfxnx5FhoCav8QAvD_BwE
Hi,
In electrophoresis stage, you may want to set longer time with lower voltage to get rid of the traces or dimers.
Hello,
I suggest that you adjust the primer concentration in your PCR mix since too high a concentration will result in dimer formation and check your template purity. Hope this helps!
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