We had a somewhat similar problem a few years ago and we were able
to get sequence by altering the thermocycling conditions. My reasoning was
- if it was generated by PCR, then it must be able to be sequenced by PCR.
We went back and checked to make sure that we could PCR the piece from the
original lambda DNA, then used those identical thermocycling conditions
for the sequencing reaction. I don't remember the exact conditions, but
I'm sure there was a hot start and all steps were for longer times. It
wasn't pretty, but it worked.
Scottie
>At 1:03 PM -0400 8/11/99, Thomas J. Stelick wrote:
>We are trying to sequence a cloned PCR product for a customer but we are
>having a problems. The pcr product has a 24 base inverted repeat at the
>ends which is causing to fold on itself and the sequence can not get
>through this structure. We have tried DMSO but no difference is seen.
>Does anyone have any suggestions on how to get through it.
>
>Tom Stelick
>TJS11@cornell.edu
>BioResource Center, Cornell University
>157 biotech bldg.
>Ithaca, NY 14853
>(607)254-4857
>http://brcweb.bio.cornell.edu