cloning the wrong way?
hzhen at freeuk.com
Wed Feb 16 20:52:47 EST 2000
"Dr. Duncan Clark" wrote:
> Verify the correct insert by PCR or restriction map then sequence. The
> only way you will clone it in the opposite direction is to put it in a
> vector such that there is no promoter in the direction you want to clone
> it or that the promoter is totally repressed.
There is another way. If the purpose of the cloning is to express that
particular protein, you could clone it into a T7 promoter-based expression
plasmid (say, pET22). No protein should be produce without T7 RNA
polymerase, therefore you should not have any problem with the cloning bit.
The problem then arise as to how to express the protein when it is toxic.
There are pLysE cells that may be effective at suppressing protein
expression in T7 system (but the cells lyse easily I think). An alternative
approach is to use cells that do not have T7 RNA polymerase (say BL21
without the DE3 bit) and induce protein expression by infecting with phage
containing T7 RNA polymerase. Novagen used to sell such phages but I'm not
sure if they still do.
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