Need: protocol for direct sequencing of PCR product

Adrian Jenkins ajenkins at nibsc.ac.uk
Thu Mar 21 06:37:41 EST 2002


Hi Koren

Sequencing from PCR Products

Equipment us an ABI 377 36 lane antique, but still works well!

I routinely sequence  PCR products from ~230 bp to over 1,500 bp in length.

After the PCR/visualisation on a gel, I run the remaining samples through the
Qiagen PCR Clean up product, and resuspend in ~30 ul of water.

Initially, I use ~3 ul per reaction/primer but can add less some times.

Purification of Sequeinig reactions is normally via Ethanol precipitation, or
the DyEx dye removal kits, also from Qiagen

Hope this helps.

Adrian

"Spence, Koren R" wrote:

> Does anyone know where I can find a protocol for direct sequencing from PCR
> product?
>
> We have been trying (unsuccessfully) to sequence PCR product on our MJ
> BaseStation.  We have been using Qiagen PCR cleanup kits and Sequencing
> reaction clean up kits, but even still, nothing is working.  Any information
> or advice would be greatly appreciated!
>
> Thank you.
> Koren Spence
> Molecular Biologist
> Bedford Institute of Oceanography
>
> ---

--

Enjoy....

Adrian


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     NATIONAL INSTITUTE FOR BIOLOGICAL STANDARDS AND CONTROL

     Adrian Jenkins          Molecular Virology Group
                             Division of Retrovirology
                             e-mail     ajenkins at nibsc.ac.uk
                             http://www.nibsc.ac.uk/

     "No plan survives contact with the enemy"  von Moltke

     "It's not the rather fast pace of life that frightens me,
     But rather the sudden stop at the end."
                                                 Anon


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