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annealing oligos

Malay curiouser at ccmb.ap.nic.in
Thu Jan 27 07:29:13 EST 2000


I am also interested in annealing oligos (adapters for cloning). Can you
please clarify how much salt you add while annealing?? I guess you need to
cool the oligos slowly, may be keeping them in hot water and let the water
cool to room temperature normally. I'd appreciate if anyone has any better
idea.

Malay

> 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?
>

Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A

Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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curiouser at ccmb.ap.nic.in



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