universal primer can take over when the forward primer is used up; (3) since the annealing temperature of the fluorescent dye-labeled M13(-21) primer is only 53°C, the last eight PCR cycles ...

Primer 1 contains a universal sequence (Tag 1 ... adding the reagents in the order listed. Overlay PCR reaction with 15 μl of mineral oil to prevent evaporation during the temperature cycling.