pcr dna purification for sequencing

[Justin Hopkins]

> > what is the best method of purification of pcr products for sequencing?
> > could you perhaps sketch the protocol briefly?
> i would not purify the pcr product, since in your pcr reaction mix there
> are only things that you need also for sequencing-pcr, like taq,
> template and nucleotides.

Actually, the PCR contains chemicals that will interfere with your sequencing
reaction.

Specifically these are 1) dNTPs which will compete with your terminators resulting
in a weaker or invisble ladder and 2) the PCR primers which compete with your
seqing primer in the seq reaction.

I have found the fasted and mosted reliable quality producing protocol is to
elimate these two chemicals by use of enzymes. dNTPs can be digested with shrimp
alkaline phosphatase and the primers with exonucleasase one. This digestion can be
done after cycling, by adding both enzymes to the PCR and incubating for 15' at 37
degrees. the enzymes are then heat inactivated at 80 degrees for 15'. Your
template is ready.

USB sells these two enzymes as a kit, suprisingly enough called "PCR Product
Pre-Sequencing Kit"

Justin Hopkins
Cancer Genetics Laboratory
8th Floor, Guy's Tower
Guy's Hospital
London SE19RT