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.
I don't know whther this was a typo, but PstI leaves a 3' overhang,
BamHI leaves a 5' overhang. Treatment of both with Klenow plus dNTPs will
fill in the Bam site and trim the Pst site. This has rarely failed for us.
Good luck,
Pam Norton
--
Pamela A. Norton, Ph.D. Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107 p_norton at lac.jci.tju.edu
