Primers annealing protocol

Yvon CAVALOC cavaloc at sun-recomgen.univ-rennes1.fr
Fri Mar 28 07:06:12 EST 1997


Pascal Mertens wrote:
> 
> 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
> 
> MAX

I guess that the addition of "some" salt would be a good thing to do.
What I did was to denaturate the oligos in TE, then I added (at 95
degrees) 200 mM NaCl and let the temperature fall down slowly to 45
degrees.

Yvon



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