pcr dna purification for sequencing
clarosa at biocomp.unl.edu
Fri Aug 4 13:52:42 EST 2000
Justin Hopkins wrote:
> > > 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
> 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"
I am forwarding your message to my old sequencing boss. however they
have found ethanol precipitation to work well....see the previous posted
web page for the protocols
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