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annealing oligos

Colin Rasmussen colin at pombe.usask.ca
Thu Jan 27 11:12:52 EST 2000

jen_the_glick at my-deja.com wrote:

> Hi.   Have two 40 bp, complimentary oligos that I am annealing to one
> another.  They have restriction sites on the ends, so after annealing I
> am cutting with the enzymes, heat-inactivating the enzymes at 65 C for 20
> minutes and then ligating.  I am heat-inactivating the enzymes because
> resolving them on a gel and gel purifying them seems awfully difficult
> for DNA this small.
> The annealing protocol I am using is mixing them in equimolar amounts in
> the presence of a little bit of salt, heating to 90 C in a PCR machine,
> then cooling at a rate of 1 degree/min.
> So when I heat-inactivate my restriction enzymes, should I be cooling
> very slowly, as I did before?  Otherwise, might I be undoing the good I
> did in the initial annealing reaction?

You might want to do that.  Alternatively, why not phenol-chloroform extract
to get rid of the enzymes.

Colin Rasmussen

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