NO expression in pKK223-3 vector in DH10B Why ? - in DH10B NO expression in pKK223-3 vector in DH10B Why ? (Dec/16/2008 )

[size="3"]Hi all,
I did not get expression for my gene in pKK223-3 vector when I transformed into DH10B and TOP10 because my friend told me he got on expression in some Host cells 3 years ago , But why a did not in spite of I repeated my experiment twice
Please any answers to solve this problem ?
Thanks
David

details, details and details. It is in the details. That is why molecular biology is so troubled.

Is the plasmid correct? Is the plasmid that you have received the right one, and without mutation.

Is "some Host cells" that your friend use, DH10B? Are you using the same strain to express the protein? DH10B is a cloning strain. Did he instead use BL21, an E. coli expression strain? Might your plasmid be expressing a toxic protein (BL21(DE3)-pLysS or C41(DE3)) or has poor codon usage (Rosetta strain).

Did you do the exact same thing? Same growth media, oxygen availability (ie shaking speed, bevelled flask, flask size, media volume), incubation temperature, same induction duration, same induction temperature, same amount of IPTG, same protocol?

or worshiped the same molecular biology gods and make the same sacrifices.

details, details and details. It is in the details. That is why molecular biology is so troubled.

Is the plasmid correct? Is the plasmid that you have received the right one, and without mutation.

Is "some Host cells" that your friend use, DH10B? Are you using the same strain to express the protein? DH10B is a cloning strain. Did he instead use BL21, an E. coli expression strain? Might your plasmid be expressing a toxic protein (BL21(DE3)-pLysS or C41(DE3)) or has poor codon usage (Rosetta strain).

Did you do the exact same thing? Same growth media, oxygen availability (ie shaking speed, bevelled flask, flask size, media volume), incubation temperature, same induction duration, same induction temperature, same amount of IPTG, same protocol?

or worshiped the same molecular biology gods and make the same sacrifices.