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:


>ChenHA 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.

Mike


Michael Sullivan
US Dairy Forage Research Center
Madison WI


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