TAE vs TBE and borate in ligations

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue Sep 26 08:59:54 EST 2000


>"Michael L. Sullivan" wrote:
>>
>> I had heard that borate, present in TBE, can be inhibitory to some enzymes
>> (eg. DNA ligase), so that if using DNA directly out of low melt agarose, it
>> was better to use TAE.  My own experience was that ligations still worked
>> OK in the presence of TBE, though.
>>
>
>Interesting.  My own experience is that borate do inhibit ligation.
>Could you confirm that your ligation is done in TBE, and not after
>purification which removed the TBE?  And have done a comparison
>between TAE and TBE? I would be interested to know.
>

My expereince doing ligations with fragments out of low melt agarose (run
in TBE) is somewhat limited.  However, when I have done them, I've
generally had a reasonable amount of DNA on the gel such that in the
ligation reaction I used only a relatively small amount of TBE containing
gel, a few microliters (already diluted 3 fold with water) in a 20
micorliter reaction-- probably only 2-5 mM final in borate.  I did have a
collegue who claimed to do ligations out of low melt gels all the time
without any problem, though.  Of course, I don't have any hard data from
him as far as his reaction condition, etc.

I suppose one could simple add borate directly to a ligation and see where
it would become a problem.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


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