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Need: protocol for direct sequencing of PCR product

Chris Spolsky spolsky at acnatsci.org
Thu Mar 21 11:31:08 EST 2002


Hi Koren,

We find that gel purifying the PCR products makes a big difference in
quality of sequence we obtain. After PCR, the product is run through an
agarose gel in TAE, product band cut out, and then taken through a gel
extraction kit (Qiaex or Invitrogen's Concert kit). We do one additional
step after the gel extraction, and that is to do a final EtOH
precipitation, redissolve in 25 ul water, and use 2 - 4 ul per sequencing
reaction. To clean up the seq. reactions, we again EtOH ppte (with glycogen
to help pptn). We sequence on a Beckman CEQ2000 capillary machine. It's a
good bit more work, but is worth it for sequence quality.

If you don't want to gel purify, you might try using different PCR
purification kits; sometimes different ones work better for different labs.

Hope that helps,
Chris Spolsky

At 08:36 PM 3/20/02 -0000, you 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
>
>---
>
>

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