Best way of purifying PCR products for sequencing

John jjmcq at atl.mediaone.net
Sat Mar 10 09:58:18 EST 2001


We use a Qiagen PCR cleanup kit and then elute with water.  It takes 5 min.
max. and very reliable.  We've done over 500 of these and now we don't even
check on a gel after.  We then dilute the eluent 1:10 and run a seq. rxn
with 10ul of that, 3 ul of Dye mix and 2ul of primer.
Over 5,000 reactions done this way in the last 2 years.

John.

John R. McQuiston
CDC Atlanta, GA





"Y.F.LEUNG" <yfleung at cuhk.edu.hk> wrote in message
news:3A933BAE.A7DBAE2 at cuhk.edu.hk...
> We use Shrimp Alkaline Phosphatase and Exonuclease I to remove the extra
> nucleotides and primers, then heat inactivate these enzymes and add the
> sequencing primer back. In general we can get very good result by this
simple
> method
>
> Y.F.LEUNG
>
> Patrick Lynch wrote:
>
> > I haven't sequenced PCR products for a while - using an ABI-377.  What's
the
> > best way of purifying the PCR products before the sequencing reaction?
DNA
> > binding resins or gel filtration?
> >
> > Thanks
> >
> > Patrick
> >
> > ---
>
> --
> Y.F.LEUNG
> Department of Ophthalmology and Visual Sciences
> Chinese University of Hong Kong
> 3/F Hong Kong Eye Hospital
> 147K Argyle Street, Hong Kong
> Tel: (852) 27623152 Fax: (852) 27159490
> email: leungyukfai at hotmail.com, yfleung at cuhk.edu.hk
> URL: http://i.am/yfleung (My functional genomics home)
>
>







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