Blunt ending??

Hiranya Roychowdhury hroychow at NMSU.EDU
Mon Jun 8 16:39:55 EST 1998

At 04:39 PM 6/8/98 GMT, jalali at wrote:
>I need to blunt end a linearized plasmid with a 5'
>overhang of 2 bases, as well as a fragment of DNA with a 5' overhang.
>The strategy I'm considering at the moment is digestion with Mung-bean
>nuclease. Any thoughts?
>Also, assuming this strategy is okay, will I need to kinase, so that I can
>ligate afterwards.
>Any info would be greatly appreciated.
>Farid Jalali
>Mushroom Research Group
>University of Toronto at Mississauga
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No No! Use Klenow and dNTP's instead. The MB nuclease is tricky and you may
end up chewing into the piece. The protocol is simple and quick (15-20 min)
and you can find it in Maniatis or the Red Book. For 3' overhangs, use T4
DNA polymerase. 
No need to kinase the plasmid after polishing.

Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at

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