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Beatrice Seguin beatrice.seguin at utoronto.ca
Sat Apr 6 16:30:42 EST 2002


----- Original Message -----
From: ""Michael L. Sullivan"" <mlsulliv at facstaff.wisc.edu>
To: <methods at hgmp.mrc.ac.uk>
Sent: Friday, April 05, 2002 4:10 PM
Subject: Re: Orientation of a PCR product in a T-vector


> 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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