Blunt ended ligation

Tony Hodge tph at mrc-lmb.cam.ac.uk
Tue Jan 3 05:08:19 EST 1995


Subject: Blunt ended ligation
From: Shayan Sharif, ssharif at uoguelph.ca
Date: 28 Dec 1994 01:38:12 GMT
In article <3dqfi4$6jl at nermal.cs.uoguelph.ca> Shayan Sharif,
ssharif at uoguelph.ca writes:
>I have been trying to do a blunt-ended ligation, but it doesn't
seem to 
>be working. At first I checked the transformation step. With the
intact 
>pGEM3 it works just fine, but I cannot get any colony (either with
or 
>without the insert) with the ligated vector. Moreover, agarose gel 
>analysis, using supercoiled marker, shows that the DNA in ligation 
>mixture is still in linear form.


What enzyme are you blunting with??  For instance SmaI has a
reported problem with nibbling the ends making re-ligation difficult
so I use HincII for better results then often ligate overnight at
room temp.


.
Tony P Hodge
Structural Studies Division
Medical Research Council Laboratory of Molecular Biology
Hills Road
Cambridge
CB2  2QH
UK

Phone (01223)  402260

Fax     (01223)  213556



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