I am trying to clone a gene and go for protein expression. But it is not getting induced . Although I checked my insert by digestion and then by sequencing before doing transformation in expression hosts but still no results . please suggest me
you've checked your insert, have you checked your vector to make sure its what you think it is? If not I would recommend you sequence your vector to make sure its what you think it is and also did you sequence your completed construct to make sure that your insert is in frame with the vector?
Hi there,
There are many parameters to consider to try to improve your expression system:
induction temperature, OD of induction, concentration of inducer, length of induction, host genotype, nature of the tag, position of the tag aso. It might also be that your general strategy (plasmid, inducer, host) is not appropriate for some obscure reasons therefore you might also consider different plasmid constructs for different expression approaches.
hii Victoria Wu
yes i had sequenced the construct and checked that it is in frame with vector
but i had not done sequencing of vector. I will do it and check it
Hi Sarabjeet, I don't know how many times I have been burned by a vector turning out to be something other than what I was told. I always sequence the vector now.
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