Nested PCR is basically two sequential rounds of conventional PCR, where the second, nested, set of primers are designed to regions within the amplicon of the first round, and the PCR product from the first round is used as a template in the second round.
Nested PCR is exquisitely sensitive and the other side of that coin is that avoidance of cross-contamination between samples is critical.
Moreover, since the template is already amplified DNA (amplified during the first round), a smaller template volume is normally used for the second (nested) round. The cycling parameters can be different, and should correspond to the annealing temperatures of the primer sets and the size of the nested round amplicon.
Agree with Marcus.Only 1ul template should be fine to be used in 2nd round of PCR. You can also increase annealing temp. to make it more specific.
shefali
I agree with both of you, just remember that first set of your primers have to be very specific, also would be good to check on the gel your first product.
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