Scott Coutts schrieb:
> 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.
>> Recently, I was told that for every protein expression experiment, I
> should re-transform my BL21s with a plasmid prep from DH5a, because they
> will lose the plasmid or will not express as well when BL21 is kept in
> the freezer. So they're suggesting that I do a transformation every time
> I want to express protein... is the the normal way of doing things?
I would consider this as normal. I never keep transformed BL21...
I transform it when i need it - do a selection plate and keep this at 4°C.
You can then pick a colony when you need it . The plate is usable for more
than a week....
>>> If they do lose the plasmid from storage in glycerol stocks - how does
> this occur? What does the glycerol do?
I kept some of my transformed BL21 in glycerol in the past - but i stopt
doing this because it is much faster to just keept the Miniprep and
transform when you need it....
I never encountered problems with my stocks in the past - but it is faster
the other way (electroporation)
>>> Also, does anybody have any explanation for the differences that can be
> see in proein expression amounts when BL21s are transformed and screened
> for expression? For example, say I clone something, do a plasmid prep
> and sequence it - everything's fine. Then I take some of our competent
> cells, which have been prepared from a broth originating from a single
> colony. Transforming them, growing them and inducing them shows they
> they express the protein - but some of them will have almost
> undetectable (by eye on a gel) amounts, whereas others will produce huge
> quantities... it's the same strain and the same plasmid in each case... ?
I never saw too big differences in the expression levels - smaller
differences are normal - i have no idea why this is the case - i guess
nobody really knows this.
Did you incubate the transformed bacteria in non-selective media directly
after transformation? this seems to be necessary.
greetings
Phil
>>> Thanks for any advice,
>> --
> Scott J. Coutts
> ------------------------------------------------------------
> Bacterial Pathogenesis Research Group
> Monash University, Australia
> Phone: +61 3 9905 4838
> Email: scott.coutts at med.monash.edu.au> ------------------------------------------------------------
