Help for cloning

R.A. Hastings RAH22 at
Mon Jun 22 14:11:58 EST 1998


I usually phosphatase vector prior to any ligation, I know most people
don't phosphatase double digested vector, but it can't hurt can it? You
also said you were concerned that the expressed protein could be toxic.
As I'm sure you know,  pRSET vectors are leaky and will express small
amounts of recombinant proteins, but only in bacterial strains which
express the T7 polymerase gene. Are you cloning the ligated recombinant
plasmid straight into BL21s? If so, I'd recommend against it.

Good luck,
Cardiovascular Research Institute
Leicester Royal Infirmary

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