I cloned a protein using a house made plasmid and transformed it into Rosetta2(DE3)pLysS (due to its rare codon properties). When I finished cloning I was able to express protein but after 2 yrs when I went back to the same stock and tried expression again it didn't work. I don't understand why?
Hi, did you stocked the transformed Rosetta or only the plasmid and use it to transform fresh Rosetta?
I will suggest you always to used new bacteria add re-transform, specialy after two years. Also check the antibiotic concentrations. Hope it helps
Greetings,
As Ferdinando said, you should be using plasmid to tansform fresh bacteria, so you get optimum results. However, if that is not a option, run minipreps to isolate your plasmid and start again. In adition of an antibiotic concentration error, these kind of problems might arise when the concentration of glycerol is not enough, so your bacteria dies because the formation of ice crystals.
Best regards.
hello,
some times it usually happens but need not to worry, you need to check the problem step wise
1) check the cells have the inserted gene?
2) check the quality of your chemicals (always use freshly made ones) and their concentrations.
3) Try to transform the gene into new cells and express.
best regards.
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