pcr product cleanup problem

Philipp Wechner philipp.wechner at uibk.ac.at
Thu Dec 12 10:54:18 EST 2002


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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