pcr product cleanup problem
rfwhittier at hotmail.com
Fri Dec 13 01:03:33 EST 2002
Sehr gefreut, wenn diese Methode auch Ihnen nuetzlich wird.
I have not noticed any problems, but neither have I done any carefuly
A/B side-by-side comparisons with purified PCR products. Assuming that
the PCR products are long enough, I routinely get more than 600 nt of
quality sequence data after cycle sequencing and capillary electrophoresis
on the MegaBACE instrument.
Your question sent me back to the kit manual to make sure I wasn't
misrepresenting anything. Actually, it turns out that it is a USB
kit that is distributed by Amersham Biosciences. The back of the kit
mentions coverage by US patents 5,756,285 and 5,741,676, as well as a
patent pending for ExoSAP-IT (i.e., the kit). I assume that this is why
other companies are not marketing similar kits.
>Your method sounds intersting - how good ar the results? Any problems with
>the methods and the outcoming sequences? how long are the sequences after
>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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