What is the best way to purify plasmids for DNA sequencing?
Plasmid templates for use in DNA sequencing may be isolated by a number of different methods from a standard alkaline lysis preparation to any number of commercially available columns and matrices. The choice will depend on cost and convenience to the user. Regardless of the template isolation procedure used, for successful DNA sequencing, the resulting template must be free of large amounts of residual detergent, salt, polyethylene glycol, and ethanol. These compounds can inhibit DNA polymerases. Avoidance of “boiling detergent” preps, and careful washing and drying of DNA pellets should yield template that produces good quality sequence data. What about PCR product templates? Both residual amplification primers and unincorporated nucleotides must to be removed from PCR product preparations prior to sequencing. Residual primers can produce spurious ladders when using internal labeling protocols or compete with the sequencing primer for extension in primer labeling protocols. Unincorpo
