Identification and Molecular Cloning of RoTat VSG Gene of Trypanosoma evansi of camel in India

The present study was carried out to isolate the Rode Trypanozoon Antigen Type VSG (RoTat VSG) gene of Trypanosoma evansi using PCR and cloning of the gene. The desired amplicons of RoTat VSG gene from genome DNA of T. evansi was successfully amplified by PCR using gene specific primers. Amplified PCR product was identified on the basis of size of the RoTat VSG gene using 25mM mgcl₂ and at annealing temperature of 51°C. For cloning the purified DNA fragment was ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid was identified on the basis of white/blue colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinant was done by Restriction Enzyme digestion of plasmid DNAs using EcoRI and confirmed on the basis of gene size, i. e 1450 bp for RoTat VSG gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.