Orientation of a PCR product in a T-vector
Leman at Leman.org
Fri Apr 5 13:21:17 EST 2002
On Fri, 05 Apr 2002 13:59:06 +0100, Bertrand Collet
<b.collet at abdn.ac.uk> wrote:
>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) ?
There is no good reason for a TA vector to discriminate against one
orientation in favor of the other. The only case where I can see a
reason for such discrimination is if you clone something to be
expressed in E. Coli and the reverse clone produces a toxic protein.
AFAIK, your cloned gene is not supposed to be expressed with the TA
system. What kind of numbers are you talking about? 3 out of 3 is not
equal 100%, even 9 out of 10 is not really 90%.
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