pcr product cleanup problem

Robert Whittier rfwhittier at hotmail.com
Thu Dec 5 21:56:53 EST 2002


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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