Preparation of Template for Sequencing

Louis Geller demode101 at mail.earthlink.net
Thu Jul 20 10:14:40 EST 2000


Marcus,
In the past, I have used Qiagen's Qiaex II resin-based gel purification system.
The only problem is that some of the resin could get into your samples, if the
protocol is not done carefully.

Could there be something else wrong such as too much salt or some other
inhibitor.
If you don't need to gel-purify your samples, could you use some other
purification method.  In our lab, for PCR products, we do Exonuclease I / Shrimp
Alkaline Phosphatase treatment to degrade primers and inactivate dNTPs.

Louis

"Pezzolesi, Marcus" wrote:

> We have recently been having problems with our cycle sequencing and feel
> that the problem lies in the preparation of DNA template for sequencing.  In
> the past, we were having quite a bit of success with cutting bands from
> agarose and then using Qiagens Qiaquick spin columns, but this is no longer
> working well. We have also tried PEG precipitation, but also with mixed
> results.
>
> Can anyone offer a protocol which has proven to work well?
>
> Thanks.
>
> ---








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