Re: [ccp4bb] problem in transformation of pqe 30 clone

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: side chain angle differences between monomers
From: iulek {- at -} INTERPONTA {- dot -} COM {- dot -} BR iulek {- at -} INTERPONTA {- dot -} COM {- dot -} BR
Date: 2009-05-04

Next message:
Subject: Charge vs. pH plot Isoelectric titration curve ascii plotter
From: Paul Smith paul {- at -} PALADINSCIENTIFIC {- dot -} COM
Date: 2009-05-04

Subject: Re: problem in transformation of pqe 30 clone
From: Raji Edayathumangalam raji {- at -} BRANDEIS {- dot -} EDU
Date: 2009-05-04

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, bas@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.

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Next message:
Subject: Charge vs. pH plot Isoelectric titration curve ascii plotter
From: Paul Smith paul {- at -} PALADINSCIENTIFIC {- dot -} COM
Date: 2009-05-04