T4-pol PstI/Exo EcoRI/fill in

NJ njuni at poppy.ocn.ne.jp
Tue Oct 30 05:52:34 EST 2001


As you say, it would be a good idea to chose T4 pol to blunt 5'- and 3'-
protruding ends simultaneously.

I have some suggestions.
Since T4 pol has strong 3'->5' exo activity as compared to 5'->3' pol
activity, it easily over-digests DNA ends, yielding 5' protrusion.
To avoid this, 
1) Do not add too much T4 pol (1-2 units of T4 pol would be enough for 0.2-5
ug of DNA).
2) Incubate at low temperatures (e.g. 12C for 15 min. At this temp, the pol
and exo activities of T4 pol are balanced. Incubation at r.t would also work
fair). If you prefer to incubate at 37C (although I would not recommend),
incubation time should be very short, and subsequently, T4 pol should be
immediately inactivated by adding EDTA or phenol (but not by heat).
3) Add dNTPs at high conc (more than 0.1 mM each).

Hope this helps.
--
NJ


in article 3BDE6248.5AEAFD1D at gmx.net, Ricky Boernke at ricky_boernke at gmx.net
wrote on 10/30/01 5:18 PM:

> Dear all,
> 
> I would like to remove all restriction sites but the HindIII site from
> the pUC18
> polylinker region. Therefore I would like to cut the vector with
> PstI/EcoRI and
> subsequently blunt the ends and religate. To obtain blunt ends I was
> thinking of
> using T4-pol since it should nibble back the PstI overhang and fill in
> the EcoRI
> site, actually within the same reaction. Well, I am always a bit
> confused about the
> exo and pol activities of T4-pol under different conditions so I am not
> quite sure
> whether this will work. Or will I have to do the blunting in seperate
> reactions?
> Any hints appreciated.
> 
> Ricky




More information about the Methods mailing list