Orientation of a PCR product in a T-vector

[AGHunt]

<< Hi everyone,

I was screening colonies carrying a PCR product cloned into a T vector by PCR,
I was surprised to see that 90 % (and 100 % in another cloning)
of the colonies had the PCR product ligated in the same orientation. I thought
there was an equiprobablity of insertion in the 2 direction
for T-cloning ?
What is the rule for this (if any) ? >>

Were you doing blue-white selection?  Occasionally, inserts can have
unanticipated in-frame start codons that can "rescue" the b-gal fragment.  This
would happen only in one orientation (the other orientation would yield white
colonies), leading to the situation you describe.  Whenever this happens here,
we screen a few blue colonies;  more than once, this suspicion has been borne
out.

Of course, if B/W selection is not being done, my post is irrelevant.

Good luck!

Arthur Hunt
Dept. of Agronomy
University of Kentucky