reproducing protein expression

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central dog-maaaa
central dog-maaaa's picture
reproducing protein expression

Hi
Iam engaged in expressing a gene of gram positive orgin in E.coli M15 cells through pQE-30 vector. Initially i got good expression; but following trials went waste as i could not express the protein. I tried different expression conditions & checked all the reagents used, all went vein. The insert is there in the cloned cell as confirmed by PCR. What could b the possible cause?
Note: Ive expressed the protein once so it could not b toxic to the E.coli.

protoldo
protoldo's picture
 Hola, if this phenomenon is

 Hola, if this phenomenon is observed ever using frozen aliquots of first transformation, you have to transform every time. But if expression is lost by passaging in plate , it¨s good to have a frozen aliquots stock to use a new each time. Buena suerte