I have this problem. Today I used a new positive control of an isolated NC-1 strain in cell culture and the problem persists.
I performed a serial dilution test to test the sensitivity of the protocol and standardized a final DNA concentration of 20ng in the first PCR reaction, as I work with samples with low DNA concentration.
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.
Flávia Moreira da Fonseca Why do you try to increase the cycle of the first PCR? About 35 cycles?
In the gel, all the bands (corresponding to wells) are visible. I don't see any reaction which did not work, as you mention in the question.
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