pcr product cleanup problem
Mark_Dowton at uow.edu.au
Thu Dec 12 18:29:55 EST 2002
We use PEG precipitation. Similar to Qiagen-type columns, you do lose some
material, but at ca. 10c per clean-up, it beats either column or ExoISAP
Philipp Wechner wrote:
> Your method sounds intersting - how good ar the results?
> Any problems with the methods and the outcoming sequences? how long are the
> sequences after this treatment?
> Robert Whittier schrieb:
> > Lea and fellow listers,
> > Lea Lutalica posted the following message:
> > <snip>
> > >I have been using Qiagene columns and ammonacetate precipitation to clean
> > >my 900bp pcr product before sequencing and I lose almost 70% of initial
> > >DNA. <snip>
> > Why purify the PCR product if your objective is simply sequencing?
> > Do you need to concentrate it? I routinely treat with Exo I and
> > Shrimp Alkaline Phosphatase (SAP), then heat inactivate these enzymes
> > prior to sequencing. The Exo I destroys your primers and the SAP
> > depletes your dNTPs. It seems a lot simpler than purifying products,
> > especially when you process many reactions simultaneously. Anyway, it
> > always works for me.
> > Robert F. Whittier
> > Senior Scientist, R&D
> > Amersham Biosciences K.K.
> > Tokyo
> > Claimer: Yes, my company sells these enzymes as well as a kit, but
> > 1) AFAIK, we do not have a monopoly on the enzymes, and
> > 2) Anybody who doesn't like ExoSAP treatment is free to comment.
> > _________________________________________________________________
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> > ---
Institute for Conservation Biology
Wollongong NSW 2522
mailto:mdowton at uow.edu.au
Centre for Evolutionary Biology & Biodiversity
Dept Evironmental Biology
Adelaide University SA 5005
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