Blunt ended ligation

Shayan Sharif ssharif at uoguelph.ca
Tue Dec 27 20:38:12 EST 1994


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.
I have tried these to sort out the problem:

1- Different molar ratios of insert:vector from 3:1 to 1:3.
2- Religating the vector without any insert.
3- Different incubation temperatures and lengths of time.
4- Comparing T4 DNA ligases supplied by NEB, Pharmacia, Gibco, and Promega
(approxiamtely 5-6 Weiss units or equivalent amount of NEB T4 ligase).
5- A range of MgCl2 and ATP final concentrations, from 50 to 100 mM
and 0.1 to 1 mM respctively.

Any suggestion is welcome. Thanks.

Shayan

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