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annealing oligos

Joseph C. Bagshaw jbagshaw at wpi.edu
Sun Jan 30 11:37:21 EST 2000


Your working way too hard.  As others have suggested, just design the
desired sticky ends into your oligos.  If you don't dephosphorylate your
cut vector you won't even need to phosphorylate your oligos.  With 40-mers
you couldn't keep 'em apart with a crowbar, even at room temp.  Just mix
your oligos in ligase buffer, add your cut vector and a pinch of ligase,
and follow your favorite ligation and transformation protocols.  Been
there, done that.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.

On Wed, 26 Jan 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?
> Thanks!
> Jen
> Sent via Deja.com http://www.deja.com/
> Before you buy.

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