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