You can use PCR primers BUT just because your primers work in PCR reactions does NOT mean they’ll work for sequencing. Removal of residual PCR primers is critical. Your primers must be designed to be compatible with our sequencing conditions.
PCR PRIMERS CAN WORK VERY WELL IN SEQUENCING, BUT THERE ARE LIMITATIONS:
PCR is exponential, sequencing is not.
With two primers replicating opposite strands, a PCR reaction is exponential. Primers that are inefficient can still work for PCR. Most people amplify beyond the linear part of the reaction, meaning inefficient primers will produce as much product as efficient ones. For sequencing, this is NOT the case. Inefficient primers will give only weak bands. Also – and this is often very important – PCR will amplify even if the target DNA is only a small proportion of the DNA present. 99% of your plasmid prep may be some junk DNA, but the remaining 1% will amplify just fine. However, it will not sequence.
PCR reactions can be tailored to the primer; sequencing reactions cannot.
You probably adjust your PCR conditions to improve yield and specificity. The annealing temperature and Mg++ concentration are usually critical, but adjustments in extension and denaturation temps and times can be helpful as well. Unfortunately, we simply cannot do that in a Sequencing Core. Samples are processed 96 at a time, and we have to use consensus conditions throughout. Please study our guidelines for the design of sequencing primers; they were written with these facts in mind.
PCR reactions actually can use a mismatched priming site; sequencing rarely can.
A primer may start out mismatched against the template, but if even ONE primer manages to anneal, even briefly, the extension product will now have a perfect match and will amplify extremely well during subsequent cycles. Sequencing reactions have no such benefit. With only one primer copying one strand, the reaction will ALWAYS be less efficient for a mismatched primer.
You can use your PCR primers to sequence PCR reactions, BUT there are a few caveats: