have u tried add 0.5% -1% glucose in ur LB media (after autoclaved),
which may reduce some leaky expression of T7?
On 3=D4=C228=C8=D5, =C9=CF=CE=E76=CA=B130=B7=D6, cv2... from columbia.edu wrote:
> 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 L=
B).
>> 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
