direct sequencing of PCR products
rsteinbe at etowah.uokhsc.edu
Tue Jul 22 11:21:26 EST 1997
J Atkinson wrote:
> Any hints for successful sequencing of PCR products?
> I want to sequence a 100bp PCR product without cloning. I have been
> told a method exists which uses DMSO in all solutions, anyone know
> anymore. How small can the primer be? Any tips to get sequence as
> close to the primer as possible.
I held off replying because we were just about to try the new Amersham
33P terminator kit for just this sort of application (no affiliation).
We just got our first results and they look very good. Rather than
follow the Amersham protocol for pretreating the PCR products, we ran
the products out on a 2% NuSieve GTG agarose gel (in TBE), which is a
low melting gel suitable for small DNAs, stained with 0.5 ug/ml ethidium
bromide, cut out the bands, and melted them at 65oC to add to the
sequencing cocktail. About 1 to 2 ul of the DNA (~20-50 ng) was
sufficient to see a clear sequence on an overnight autoradiographic
exposure after 20 cycles of sequencing, but we think 30 cycles might be
better. We were able to read from the first nucleotide after the primer
for two (of four) primers we tested (we think the problem with the
others was with inadequate mixing of the acetate added to the lower gel
buffer reservoir rather than with the sequencing reactions themselves),
and the sequences looked very clean.
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