opinions: plasmid loss, protein expression differences

Mark Farrar micmdf at leeds.ac.uk
Wed Jun 19 05:44:57 EST 2002



Scott Coutts wrote:

> Hi Everyone,
>
> I'm a PhD student that's been working in the lab now for 3 years
> (including honours). So I know what I'm doing, but I dont have a _lot_
> of experience. Anyhow, when I do protein expression work in E. coli, I
> first transform, then maintain a glycerol stock at -70oC of DH5a that
> contains my plasmid (usually pET.15b). For the expression part, I
> transform BL21. My question is:
>
> How many people here, if any, store their BL21 strains for protein
> expression in glycerol stocks? I have always done this. Whenever I need
> to do the expression, I take some BL21 from the glycerol, grow it on a
> plate and pick a colony for a culture for expression.
>

I've never had a problem using glycerol stocks of BL21s with pET30b. I seem
to remember reading somewhere that plasmid loss can be reduced by using a low
% of glycerol eg 10%. Has anyone else come across this?

Mark





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