help: why NsiI enzyme digestion site can't be folled in? how can I do that?

David Micklem drm21 at mole.bio.cam.ac.uk
Sun Sep 22 11:24:44 EST 1996


In article <32451E70.35EB at biotec.jouy.inra.fr>, yuyx at BIOTEC.JOUY.INRA.FR
(yuyanxing) wrote:

>I heard from someone else that the NsiI enzyme digestion site can't be
>filled in. Is that right? If so ,How can I find another way to let it
>become blunt end? with T4 DNA polymerase? thank you for your help.
>
>my e-mail;yuyx at biotec.jouy.inra.fr

Thats right.  Its a 3' overhang so you  can't fill it in.  However, you
CAN chew back the overhang to give a nice blunt end.  
I use T4 DNA pol for this (although I think Klenow does actually work). 
Add dNTPs to your digest (to about 100uM) and a tiny tiny bit of T4 DNA pol.  
Leave at room temperature for a few-30minutes (not too long or the
nucleotides run out and the polymerase munches up your DNA...)

Good luck,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
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