t4 polymerase

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.

Hi,
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
Vienna
AUSTRIA




More information about the Methods mailing list