For sanger sequencing reaction with BigDye terminator mix, I will need to purify the excess unincorporated terminators, primers, dNTPs from my PCR product. But in times when I won't be able to purify immediately, how do I store the unpurified PCR product? Is it acceptable to simply store them in -20C until I am ready to purify? Will the terminators and other components of the BigDye reaction mix interfere with the PCR product?
The pcr amplification has NTPs but no terminators. It is the sequencing reaction that uses terminators. It will be ok to store amplified pcr product for long times at -20 or periods of less than 7 days 4c will be ok. There may be some generation of primer dimers at 4c but if your pcr purification gets rid of all primer and primer dimers then you should have no problem with storage. You will have many millions of copies of your amplimer and losing a few to freeze thaw breakage will not have a significant effect on the pcr template
Paul Rutland My reaction mix already has the BigDye mix with terminators, primers and the DNA template. Then I run the reaction mix in the thermocycler for the incorporation of the terminators into the PCR amplicons. My question was regarding this step, whether I could store these unpurified sequencing products indefinitely before I proceed onto the purification step.
Read the protocol that came with the BigDye kit. They recommend keeping the plate on ice or storing at 4C, but not indefinitely.
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