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problem in cloning - (Apr/25/2013 )

I tried to over-express GST-tagged protein in PET41a(+) in BL-21 strain,but couldn't get the protein. The cloning and overexpression was done by other person -the gene is cloned in pGEMT VECTOR ,gel eluted in large scale and ligated to the PET41a(+), he got the sequencing results and overexpressed the protein, he didn't keep the plasmid alone instead in the BL-21 STRAIN ,THIS IS IN THE -80 FOR ABOUT 1 YEAR. I have used this for the overexpression -but didn't work to give protein. To ligate , i got the cloned pGEMTplasmid in DH5 alpha and did restriction -got the gene; but while doing PCR no amplification.please throw some light to get start with my doctorate ......,


Re-sequence the plasmid to make sure that the gene is there. Once that is done, assuming the plasmid is fine - try re-transforming the DNA into the BL21 and see how that goes. Some plasmids don't store well in the expression strains.


I have heard (but not personally experienced) that there is a problem with storing BL21(DE3) strains that have a plasmid, and that you should retransform the strain rather than using a frozen stock. This never made sense to me, but perhaps it is true.