pcr dna purification for sequencing

Marc Greener mgreener at hgmp.mrc.ac.uk
Fri Aug 4 12:42:37 EST 2000


couldn't have answered better myself. even WITH butanol :-)

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
> 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

--
Marc Greener
Molecular Neuroscience Laboratory,
Division of Medical and Molecular Genetics,
8th Floor, Guy's Tower,
Guy's Hospital,
London SE1 9RT
England

Tel: +44-(0)207-9552510
Fax: +44-(0)207-9554644







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