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?
Thanks!
Jen
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