Is it really necessary to purify the PCR product before sequencing?
Yes. PCR product contains non-incorporated dNTP and primers that must be removed before sequencing (dNTP from the amplification reaction changes the dNTP/ddNTP concentration ratio in the sequenation reaction). In cases the PCR product is isolated from the agarose gel, it also contains excessive salts present in the electrophoretic buffer. Some commercial kits can be used, which operate on various principles: DNA bond to a little silicate membrane (for example, QIAquick PCR Purification Kit), separation of salts, dNTP and primers based on molecular weight using a gel (for example, CENTRI SPIN 20) or enzymatic single-step purification (for example, ExoSAP). The yield of the purification depends especially on the length of the PCR product, and the resulting concentration must be verified by means of electrophoresis – See here for more information >>.
