I am trying expressed 24 Kda protein but its not happen. Before one year ago i successfully expressed and purified but now its not expressed in E.coli BL21 (DE3) strain. i also tried various IPTG and temperature concentration.
so help me in get out of it
Is it possible that your competent cells or your vector is degraded? They can lose transfection efficiency really fast if freeze thawed. I would buy a new competent cell and try again. If no improvement, I would clone my gene into the vector once again (you can purhase this service).
Are you using a selective media? I mean for example are you using kanamycin etc to see if you are selecting correctly transfected cells? When you retransform and put on solid culture media are you getting colonies? Or no growth at all?
Is your problem not being able to get protein or not being able to grow the cells?
I m using ampicillin and when i retransform in BL21 i get colonies.
my problem is not get protein.
Do you have SP for the gene of interest, if you had please check different conditions for the expression of SP such as contituents of medium, temperature, pH etc..
are you having the previous stock of expressed protein plasmid? if you are having , then using that plasmid go for transformation. you will get your protein
As ec Sener already wrote you should start with an restriction digest of your vector. The grow of your bacteria doesn`t tell you if your insert is degraded or mutated somehow. I was facing similar problems a while ago and in the end it was a fully destroyed promoter region of my plasmid.
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