help for blunting 5' and 3' overhangs
pnorton at lac.jci.tju.edu
Thu Oct 17 13:29:06 EST 1996
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.
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.
Pamela A. Norton, Ph.D. Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107 p_norton at lac.jci.tju.edu
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