Orientation of a PCR product in a T-vector
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Fri Apr 5 16:10:14 EST 2002
At 07:11 PM 4/5/2002 +0200, you wrote:
>>Bertrand Collet wrote:
>>>I thought there was an equiprobablity of insertion in the 2 direction
>>>for T-cloning ?
>>Whatever that got expressed in one orientation is toxic to the cells.
>That's what I always thought as well.
>Now, I actually already have this problem and I need the insert in the
>correct orientation (not in T-vector but later) - does anyone have an idea
>how to prevent E. coli from producing my protein and killing itself? It's
>under a cyanobacterial promotor which is apparently leaky, at least in
>DH5a. Is my only option now to try different strains?
OK, I'm not basing this on anything other than empirical observation, but I
think that one particular orientation can be favored without the insert
necessarily being toxic (or at least lethal). That is, I've seen cases
when I've not been able to recover a particular orientation, redesigned the
cloning to be directional, and then recovered the orientation I
wanted. Perhaps the desired event is greatly unfavored by a particular
orientation, but not impossible if one can come up with a directional
cloning strategy instead of a non directional one (which IS usually
possible with PCR).
I've no doubt there are cases where an insert is so toxic it can't be
cloned in a particular direction, but I have a feeling that "the insert is
toxic" gets invoked even when it's not true.
Just my 2 cents.
US Dairy Forage Research Center
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