Primers annealing protocol

Pascal Mertens Pascal.Mertens at
Fri Mar 28 04:21:21 EST 1997

Hi there,

WE want to anneal two primers (135 bases lenght) and to clone them in a plasmid.
Here is our first assay protocol:
We mixed 5nmol of each oligo (in TE buffer) to a final volume of 111 microL.
The mix was heated for 5 min at 95 degrees, and then cooled down to 45 (1
degree per min) and then transfered on ice.

We didn't obtain any ligation product. We analyzed the annealing product
on acrylamide gel: there was a band at the same level than for the oligos
(ran separately) and a light smear upper to this band. So the annealing
does not seem to be good.
Any advices?

Thanks in advance


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

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