T/A cloning

Alex C. Jeffries a.jeffries at uts.edu.au
Sun Jan 14 21:34:51 EST 1996


On Mon, 08 Jan 1996, Martin Cann wrote;

>The wobbly T's on these vectors are notoriously unstable.  The cut >vector has a shelflife of 1-2 months and will not survive too many >freeze-thaw cycles.  Could this be it?

This is interesting, I have not heard of this before.

The problem could also be caused by small amounts of DNase contamination that is chewing off the T overhangs and resulting in blunt ligations. It may only take a very small amount of contamination to result in a number of blue colonies. I'd suggest using fresh aliquots of the reagents and maybe doing a side-by-side experiment where one assembles the ligation at the bench as per usual and also assembles one in a hood using gloves etc.

Alex

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Dr. Alex Jeffries
University of Technology, Sydney
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