Molecular cloning of adenosine transporter 1 gene of Trypanosoma evansi of Indian camel

The present study was carried out to isolate the adenosine transporter 1(TevAT1) gene of Trypanosoma evansi using PCR and cloning of the gene. The desired amplicon of TevAT1 gene from genomic 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 TevAT1 gene using 25mM MgCl2 and at annealing temperature of 49°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. 1413 bp for TevAT1 gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.