Orientation of a PCR product in a T-vector

Michael Witty mw132 at mole.bio.cam.ac.uk
Fri Apr 5 08:13:51 EST 2002


On Fri, 5 Apr 2002, Bertrand Collet wrote:

> 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) ?
> Thank in advance,
>
> Bertrand
>
> PS I ask this because I will need soon some "inverted" PCR product (the
> rare orientation in the above examples) for subcloning purpose ...

  Dear Bertrand,
               whenever I heard this before people have put it down to
magic.  If you need inverted clones, why not use restriction enzme sites
in the vector rather than redoing the PCR.  This will subject you to a
different set of "magic" rules (hopefully).  Mike.




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