Fw: Fw: sequence services

Eric DUFOUR Eric.Dufour at cmm.ki.se
Fri May 11 05:36:21 EST 2001


    Let's say you have a clean & reproducible amplification of your > 700bp
fragment (single or at least isolated & intense band on agarose gel after
electrophoresis). Then you can cut out the band from the gel and sequence it
directly from agarose (I don't have the protocol here but I can find it, it
is commonly used...). You can use the primer you use for sequencing to
amplify the sequence but it gives usually low quality sequences. You will
have better results using internal primers if you know at least a part of the
amplified sequence (usualy it is the case). If one of these possibilities
work then you can simply design new primers as long as your sequencing
progresses (to sequence more of the amplifiedc product and also to
re-sequence the parts you have already sequenced & sequence the extremities
near the primers. I've done that for a 1.2 Kb region downstream of a gene and
it worked perfectly (but it was on an haploid organism). With one
amplification you can make several sequences (around 10 at least ???), I
don't realy remember how many.
    If your gene is unique it will allow you to detect polymorphisms, if
there is too much of them it'll be unreadable and you'll need to clone your
PCR product. If your gene is in multiple copies it's safer to clone it first
unless you know that there is no or very low polymorphisms. Remember also
that intronic regions presents usualy more polymorphisms than exons
sequences.

Hope it helped
Eric


Deanne Bell wrote:

> O.K. So . . .
> If you want to sequence something >700bp you need to know both forward and
> reverse primer sites (so you can sequence from both ends)
> And you need to be able to disregard the first 50bp
> And there can be several copies of genes in any given genome
> It sounds to me like it is wiser to clone all the time
>
> So what are the conditions in which you can skip the cloning step?
>
> I'm not trying to beat a dead horse, I really am trying to learn this
> stuff.
> Thanks again
>
> Deanne Bell
> Molecular Markers Lab Technician
> USDA Agricultural Research Service
> 2021 S. Peach Ave.
> Fresno, CA 93727
> e-mail: dbell at qnis.net
> phone: 559 453-3170
> fax: 559 453-3088
>
> ---




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