Primers annealing protocol

Paul N Hengen pnh at ncifcrf.gov
Fri Mar 28 10:33:44 EST 1997


Yvon CAVALOC (cavaloc at sun-recomgen.univ-rennes1.fr) wrote:

> 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?

| 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.

Why not start with a very high conc. (10-20 mM) of both oligos in ligation
buffer?  Make 2-fold dilutions of that in ligation buffer, then heat 90 C for
10 min., followed by a slow cooling. To each tube, add 1 ul of prepared vector
and 1-2 Units of ligase. Incubate and transform 1 ul into E. coli. That should
work! I've done this several times.

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