Manuel Maria SIMON
Simon at mail.boku.ac.at
Tue Apr 18 06:19:11 EST 1995
uxa.cso.uiuc.edu (dona robinson) wrote:
> Did anyone use the 3' exonuclease activity of t4 polymerase to cut off the
> 3'overhang ends? How does it work and any percaustions needed?
> When I don't understand I ask many questions, when I do understand I ask even more.
There are some critical points using T4-Pol.
1. adjust your dNTP concentration to 200 microM. This will make the
enzyme stop at the douplex DNA. Otherwise the exo-
nuclease activity will degrade also into your doublestranded DNA. The
reaction time is some 5 to 10 min only at 37 but also at room temperature!
Do not incubate for a longer period of time.
2. In my hands killing of the enzyme by 65°C is not very complete!
If I ethanol precipitated such blunted DNA (and thereby remove dNTP)
residual T4 activity sometimes starts to shorten down my fragments.
Therefore, I either phenolize after making blunt ends with T4-pol.
Alternatively, if I do ligations, I leave the fragment in the T4 buffer,
(including dNTP) and only supplement with ATP and ligase.
hope this helps
Manuel M. SIMON PhD
Center of Applied Genetics
Univ. BOKU Vienna
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