Dear everyone, I am trying to clone a viral protein in the E. Coli BSJ strain and i am having some problems.
I start from the viral RNA carrying out a reverse transcription and PCR (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for mutations, there are no mutations. So the RT-PCR works fine. Then, I digest the cDNA and I ligate it with a pET plasmid to transform the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR) but when I sequence them I get various mutations (aprox. 2 miss-sense) on the inserted cDNA. Furthermore, these mutations are different among different transformations and even among colonies of the same plate (in the same transformation). Maybe these mutations are produced by the cell (because of the lack of mutations in the cDNA) but these E. Coli clonning strains are supposed to be "optimized" to prevent the insertion of mutations. So I have no idea about what may be the problem. I hope you could help me. Thank you. Best regards, -- --------------------------------------------------- Rubén Sánchez
