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expression problem


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#1 saher

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Posted 17 August 2011 - 07:59 AM

Hi,
i have cloned a gene in pet 222b and now wanna take expression in bl21C+. but when i give it induction with IPTG (0.5mM) O.D does not increase and i am not getting any expression. can anyone plz tell why is the o.D NOT increasing ??? Why am i not getting the expression :(

#2 saher

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Posted 17 August 2011 - 08:01 AM

my protein is of 39kDa

#3 prodes

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Posted 17 August 2011 - 08:33 AM

Hi sasher,

Do I understand correctly that you observe cell growth before induction and arrest after induction with IPTG?

If this is true, then your protein is most likely dramatically toxic to the cell. This might happen rather with enzymes like nucleases for example or some other that interferes significantly with the cell's regular functionality. For example a kinase might need to be coexpressed with a phosphatase.

If you provide more information about the protein and your expression protocol more specific suggestions may follow.




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