Blunt ending??

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Tue Jun 9 00:01:07 EST 1998


In article <6lh44n$9hp$1 at nnrp1.dejanews.com>, jalali at my-dejanews.com wrote:

> I need to blunt end a linearized plasmid with a 5'
> overhang of 2 bases, as well as a fragment of DNA with a 5' overhang.
> The strategy I'm considering at the moment is digestion with Mung-bean
> nuclease. Any thoughts?

My summary on this topic (from several years ago) may still be in
the archives but the bottom line (from net advice and from direct
comparisons by myself) is as follows;

Don't use mung bean nuclease (although there are manufacturer to
manufacturer variations so some preparations may actually work according to
theory).

For 3' overhangs use Klenow and for 5' overhangs use T4 polymerase.
Some protocols call for incubations in the absence of dNTP's to
allow nuclease activity and then further incubation with dNTP's
to allow filling in to ensure the ends are blunt.

An alternative is to use Pfu polymerase (72degC +/- dNTP's).

I've personally used Klenow, T4 and Pfu with good success.  The
Pfu protocol can be obtained from Stratagene (it is also published
in N.A.R.) and the others are in most molecular biology texts.

> Also, assuming this strategy is okay, will I need to kinase, so that I can
> ligate afterwards.

No, they all leave terminal phosphates.  If this is just an intramolecular
(re)ligation it is very efficient.

     I hope that this helps,
          Bernard
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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