short sequencing

Pascal Mertens 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.
> 

First.
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.

Second.
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
mix).

hope this helps
Pascal

-- 
Pascal Mertens
Laboratoire d'Immunologie et Microbiologie
URBM-FUNDP
61 Rue de Bruxelles
5000 Namur, BELGIUM
Phone: 32 81 724438
Fax: 32 81 724420



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