One thing to check is whether there is too much DNA in the
transformation reaction. This is sometimes a reason for failed
transformations, be it DNA from regular minipreps, PCR DNA or
ligation reactions etc.
Raji
On May 4, 2009, at 3:57 PM, b...@freesurf.fr wrote:
This story is rather puzzling indeed, and it is difficult to see
why in
case of toxicity one would have transformation problems with that one
strain in particular.
Also, I am wondering how likely it is that a combination of
toxicity and
leaky expression would lead to transformation problems in the first
place.
I have quite a bit of experience with the expression of extremely
toxic
proteins/peptides and I usually find that most of the popular E. coli
strains are quite capable of somehow getting rid of the gene or its
expression if they really need to, while retaining full antibiotic
resistance (the upshot of which is a normal transformation
efficiency but
zero expression upon induction).
My advice would be to first check very carefully for (extremely)
trivial
reasons for the failed transformation.
Best wishes, Sebastiaan Werten.
----- Original Message -----
From: Cynthia Kinsland
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, May 04, 2009 4:52 PM
Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone
Using pQE30, any E. coli is an expression host. Because it uses
the T5
promoter, you don't need an E. coli strain carrying the T7 RNA
polymerase
(so, you don't need a "DE3" strain).
As noted by Artem, you are most likely having a leaky expression
problem.
However, it is odd that DH5a will transform if this is the case
since it
does not carry the lacIq mutation. XL1-Blue does, which is why it was
suggested below.
You could try expression right in DH5a, since you have the plasmid
there.
Your transformation difficulties seem strange. Using this vector,
DH5a
should not transform any more stably than the other strains you tried.
