There's a good chance that when you heat to 65, you're de-annealing the
oligos. I also design mine such that the RE sites are made when the oligos
are annealed.
There is a message in the archives by Paul Hengen that I've used for
years--March 28, 1997. Do a search for 'primers annealing protocol'. It's
worked time and again. If you can't find it, let me know and I'll try to
re-post it.
rich
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?
>> Thanks!
>> Jen
>> Sent via Deja.com http://www.deja.com/> Before you buy.
--
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Cystic Fibrosis Research Center
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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