help for blunting 5' and 3' overhangs

[Pamela Norton]

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