Help for cloning

Dom Spinella dspinella at chugaibio.com
Wed Jun 17 14:46:49 EST 1998


> Hi, all:
> I am cloning a cDNA into His tag fusion protein vector (Invitrogen). No
> matter how I try, the target cDNA won't get into vector. The strategy
> for the cloning is very straight forward, double digested (NotI/EcoRI)
> the target cDNA and the vector as well, used 1 vector to 4 insert DNA
> molar ratio to ligate. After transformation, only a few colony showed
> up. All of them were vector shelf-ligation. I did a lot cloning before
> and never had such experience. I thought it might toxic protein. If it
> do so, what fusion protein system I could use? Any suggestion will be
> greatly appreciated.
> 
> Li

Li:
I don't think the problem is with your cloning strategy at all. It
sounds as though the fusion protein is indeed toxic to the cells --
something that's not uncommon.  If I am not mistaken, the Invitrogen
vectors allow for inducible (IPTG) expression of the fusion protein (I
presume you are dealing with a prokaryotic expression system here).  Are
you adding IPTG in the initial plating of the transformed bugs? If so,
what happens if you don't add it? The Lac operon is pretty "leaky" --
you still get some expression even without the inducer and if your
protein is very toxic, bugs won't grow even absent IPTG.  You might
switch to inducible expression systems with more tightly regulated
promoters.  The pL expression system of Invitrogen is pretty good in
this regard (trp-inducible) -- you might consider that.

Also what strain of bugs are you using for transformation?  And what is
the fusion you are trying to express?
--Dom Spinella



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