In article <53u0b0$47i at news.acns.nwu.edu>, ikekim at nwu.edu wrote:
> I am trying to modify a gene by blunting the ends of Pst1 (gives 5'
> orverhang) and BamH1 (gives 3' overhang). We have tried to blunt the ends
> by using T4 polymerase to chew up the ends of Pst1 site and fill in the
> 3' overhang of BamH1. However, we had no luck. Does anyone have any
> suggestions for us to try? Please specify the enzymes that you have used.
> You can post the answer on the net. Thank you.
>> Isaac.
Hi!
It's just the other way about: BamHI produces 5' overhangs, PstI 3' overhangs.
I have used S1 nuclease to blunt 3' overhangs, and it is good practice to
treat the DNA with Klenow enzyme as a second step. Klenow fill-in alone is
sufficient with 5' overhangs.
You can find the S1 buffer and protocol in molecular biology manuals. My
fill-in protocol is in the Klenow enzyme entry of the New England Biolabs
catalog.
S1 functions on single-stranded DNA. It will cleave double-stranded
molecules only at mismatched or single-stranded regions. Klenow polymerase
has a 3' to 5' exonuclease activity opposing polymerization, but this is a
problem only if high incubation temperatures are used. I carry out Klenow
reactions at 20 to 25 C and inactivate the enzyme by phenol:chloroform
extraction, not by heating.
Good luck.
Jose
Wellcome Trust Centre for Human Genetics
Oxford OX3 7BN, UK
Email: jose.mejia at well.ox.ac.uk
