Isolation of m16D10L parasitism gene from pUC57 for cloning into pET11a-link-NGFP vector

The purpose of this study was to isolate a mature 16D10L peptide gene sequence (m16D10L) from pUC57 in order to clone it to pET11a-link-NGFP::m16D10L vector. A specific primer pair was designed to contain XhoI and BamHI restriction sites to amplify m16D10L fragment (64 bp) that was previously cloned into pUC57. PCR optimisation steps were carried out to determine the optimum annealing temperature (TA), Mg2+ concentration, and Taq polymerase

concentration for the reaction. From this analysis, we chose 49.6oC as the best TA value, 1 mM Mg2+ and 1.75U Taq polymerase to be used in PCR reactions for gene amplification. However, faint bands were obtained resulting in the failure of cloning this gene into pGEM-T Easy vector and transforming the plasmids into E. coli JM109. Therefore, since the target gene is short, we changed our strategy to instead produce m16D10L by annealing the forward and reverse oligonucleotide fragments in a touchdown PCR reaction. We had successfully obtained m16D10L DNA fragment of ~60 bp using this strategy.