PCR product seq.

Victor A. Kim Victor.A.Kim at dartmouth.edu
Wed Nov 8 23:47:09 EST 1995


Hi!

I was wondering if anyone out there could help me with a sequencing
problem I have been constantly experiencing.  I have been trying to
directly sequence a 200 bp PCR product using New England Biolabs
thermal cycle sequencing kit.  I can read patches of the sequence
clearly, but every so often I get bands across all 4 [GATC] lanes
making those regions of the sequence unreadable.  I have already
subcloned and got a clear sequence of this PCR product, but I would
like to *directly* sequence the PCR product so I can avoid subcloning. 
The PCR product is pretty clean in my opinion [gel purified] so I don't
think it's the quality of the PCR template.  I have also tried using
nucleotide analogs such as deaza G and deaza T and all I get with those
are huge smears on the gel.  I'm thinking now that perhaps cycle
sequencing is too sensitive for PCR product sequencing?  Thanks for
your input!



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