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.
I second the reclone it recommendation.
Best,
Cynthia
On May 4, 2009, at 10:29 AM, Engin Ozkan wrote:
Have you tried M15[pREP4], which are the cells Qiagen would like you
to use? You can at least use pREP4 + your expression host, and have
repressor expression to prevent leaky expression. That can help you
get colonies of transformants.
Engin
On 5/4/09 7:04 AM, atul kumar wrote:
xl1-blue is not an expression host,since i have cloned it
successfully,i need to transform into expression host, i am able to
transform it into dh5 alfa,but not in any of expression host
From: ar...@xtals.org [mailto:ar...@xtals.org]
Sent: Mon 5/4/2009 6:32 PM
To: atul kumar
Cc: ccp4bb@jiscmail.ac.uk
Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone
Hi,
You are using a pQE vector which has a T5 promoter. T5 is a native-
like
promoter, recognized by the E. coli RNA polymerase - and this means
that
even with lac operatorsupstream there is a huge amount of leakage
in this
system. If your protein is even moderately toxic then you have
issues.
Your solutions are
1) to use cells with higher levels of lac repressor (XL1-blue for
example)
2) to re-clone this ORF under some tightly controlled promoter
Artem
>
> i have cloned my gene successfully into qiagen vector into pqe30
but i do
> transformation of this into BL21,pLys,Rosseta,C41, i dont get any
> colonies,comp cells are good other clones give good no of
colonies upon
> transformation.
> can someone help???
> thanks
> atul
>
