A Sanger sequencing reaction is run with a single primer. The following table lists the recommended amount of DNA template and primer for optimal Sanger sequencing results. Note: for plasmid DNA the ...

primer and 2 pmol of the forward primer in a final 50 μl reaction volume (Perkin-Elmer Standard PCR reaction buffer, 0.2 mM dNTPs, and 50–100 ng template DNA, 1 U AmpliTaq DNA polymerase).

Synthesize or order the template oligonucleotide designed in steps 1 and 2 and the 5′ and 3′ primer oligonucleotides (Fig. 3). Because very little primer is required for the PCR reaction ...