do not ligate at RT for O/N

Burnap Lab burnap at vms.ucc.okstate.edu
Tue Sep 19 12:24:09 EST 1995


In article <43lfht$ilk at nntp.ucs.ubc.ca>, bina at unixg.ubc.ca (rew) wrote:

> I DO NOT HAVE MUCH EXPERIENCE DOING LIGATIONS AND I HAVE TWO QUESTIONS:
> 
> 1. I DO NOT HAVE ACCESS TO A 15C INCUBATOR AND WAS WONDERING IF ANYONE
CAN TELL 
> ME IF THER IS A PROBLEM WITH DOING LIGATIONS AT RT OVERNIGHT?
> 
> 2. IS THERE A PROBLEM USING HIGH CONCENTRATIONS OF LIGASE (1-3 UNITS)IN MY 
> LIGATION REACTIONS?
> 
> THANKS FOR YOUR HELP.
> REW 

I recommend that you should set your ligation in the ref. or for 1 hr at
room temp. If you do the ligation over night at room temperture the
exonuclease activity of the T4DNA ligase increases.

Increasing the units of the ligase (besides it is expensive) it will
increase the exonuclease acitivity of the enzyme and less recombinant
clones.


Suf   Ph.D graduate Student

Dept. of Microbiology and Molecular Genetics
Oklahoma State University.



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