Transformation onto M9 Minimal Media

Hello
I have a toxic protein expressed from a T7 promoter in Pet21 D.

I'm trying to harvest the protein, but getting little yield.

I was told to do everything in minimal media to reduce any leaky
expression of T7
(I.e. to make certain there is no break down of lactose analogs found in LB).


I am using M8 MM + .4% glucose + 1mg/ML CAA + 100 ug/ML Ampicillin

Unfortunately, I am having a hard time even TRANSFORMING my plasmids
into BL21(DE3)pLysS (there should be little expression in this strain
anyway) in Minimal Media.


Even the puc 19 positive control failed.

I did the typical 30 minutes on ice, 45 sec heat shock at 42C, 2
minutes ice, 1 hour recovery (in M9 MM) .

twice this has failed (it also failed once on E.coli).

IS there any reason I should have such problems?
Please help if you can

thanks
Christal

have u tried add 0.5% -1% glucose in ur LB media (after autoclaved),
which may reduce some leaky expression of T7?

On 3ÔÂ28ÈÕ, ÉÏÎç6ʱ30·Ö, [Only registered users see links. ] wrote: