In addition to what Charlie wrote, you might try the Big Dye terminator
version 3.0 that was recently released. A few things that it will do better
than the other versions is increase the quality of the data, it is a much more
balanced reaction so the peak heights are more even, but more importantly it
tends to read through homopolymers much better than any previous version of
the big dyes. They also have big dye dGTP kit version 3, so maybe this would
be a way of reading through those inverted repeats. If this does not work,
there are a few other things that you could do, but they are much more time
and technique intensive. One way is to use transposons to insert and sequence
through. I know that in the past some research centers have used this method
to get through AT repeats/runs. Another method would be to PCR and then to
randomly break up the region of interest/inverted repeat and to ligate/insert
those random fragments into vector and then to sequence from there. Hope any
of this helps,
Eric
Charles Nicolet wrote:
> Hi:
> I have had good luck with problem templates, including an inverted
> repeat, using mixes of BigDye and the dGTP mix (3:1 BD:GTP or 3.5:0.5, just
> have to experiment) an annealing temp of 60 and an extension temp of 65. I
> also was able to get through a very difficult polyC template using the
> GIBCO/BRL Rxn Enhancer buffer D and the above conditions. Good luck!
>
> Charlie
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