Pascal.Mertens at fundp.ac.be
Tue Jun 10 06:53:11 EST 1997
In article (Dans l'article) <339CCF1D.53CF at jcu.edu.au>, James, Cook,
Universuty wrote (écrivait) :
> Hi all,
> Just hoping for some suggestions with a sequencing problem we are
> having. We are wanting to obtain the sequence of our DNA about 20bp
> from where the primer attaches.The closest we have achieved so far is
> about 40bp. Has anyone got any suggestions on how to get closer to
> the primer attachment region.
The problem is that your polymerase has to add a sufficient number of
labelled nucleotides to have your bands visible, and the closer to your
primer , the less you have!!!
So use a labelled primer.
You need to have your ddNTPs added close to your primer, so don't make any
elongation without your ddNTPs. Directly put your mix (template/oligo with
buffer with enzyme) in your four tubes (each containing one termination
hope this helps
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