I have cloned a Bacillus gene in E.Coli pET20b vector at EcoRI and HindIII sites. The vector was already having a gene cloned at the same restriction sites, therefore, before ligating the bacillus gene, the vector was digested with EcoRI(HF) and HindIII restriction enzymes so that the previously cloned gene can be kicked out of the vector. The digested (EcoRi and HindIII) PCR product was then ligated into the digested vector and positive clones were obtained which was confirmed by digestion with EcoRI (HF)and HindIII. The double digestion showed the fall off of the insert from the vector, however, the sequencing results does not correspond to the desired gene sequence. The sequence shows the gene of some E.coli strain which has similarity with the previously cloned gene as per the BLAST results. Moreover, the sequence does not have the primer sequences present and out of five recombinant plasmids sequenced, only one plasmid has the His tag sequence and others don't have. I have performed this twice and end up with the same wrong result. Is it problem of contamination or wrong primer sequence?

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