Double digest Sma1/Sac1

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Mon Jul 6 13:28:15 EST 1998


In article <kirkpat-0607981203180001 at gietz.hgen.umanitoba.ca>,
kirkpat@?cc.umanitoba.ca? (Rob Kirkpatrick) wrote:

> In article <6nqn62$pb3 at is.bbsrc.ac.uk>, "Andy Bettany"
> <andrew.bettany at bbsrc.ac.uk> wrote:
> 
> > Dear All,
> > 
> > I need to double digest a plasmid with Sac1 and Sma1. Both enzymes cut in
> > the same buffer (according to Boehringer). Can I add both enzymes and
> > incubate at 30C? Or do I need to do the
> > Sma1 first at 30C then add the Sac1 and put at 37C?
> > Thanks for your help,

> In situations like this, we'll start the digest off with Sma1 at room temp
> for an hour then add the second enzyme and put it at 37C.  Sma1 still
> works at 37C, but we like to give it that optimal head-start then it can
> finish off it's life at 37C.  Works like a charm!!!
> Rob

Okay, just to confuse matters I'll venture another opinion.
I'd suggest digesting first with SacI alone at 37degC and
then dropping the temperature and adding the SmaI.
My reason is that SmaI can nibble back at warmer temperatures
and you may not finish up with the exact ends that you
intended.  If you look back through the archives you'll
find a few posts on digests with SmaI (I believe some people
even suggest dropping the temperature to ambient or below).
     I hope one of the methods gives you what you want,
          Bernard
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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