Abstract

Abdul V. Tendulkar and Sachin Kalam Modi

Cloning is the process of producing populations of genetically-identical individuals that occur in nature when organisms such as bacteria, insects or plants reproduce asexually. Modern cloning vectors include selectable markers (most frequently antibiotic resistance markers) that allow only cells in which the vector, but not necessarily the insert, has been transformed to grow. Additionally, the cloning vectors may contain color selection markers which provide blue/white screening on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR. TOPOTA cloning provides one step cloning strategy for direct insertion of Taq polymerase amplified PCR products into a plasmid vector ligation (TOPO Cloning Reaction). In the presence of salt in TOPO Cloning reaction by mixing PCR product and TOPO vector, for transforming bacterial strain into competent cells, DH51α is supplied with the kit. Analysing transformants take 10 white colonies and culture them overnight in LB medium containing 50 µg/ml ampicillin or 50 µg/ml kanamycin. The plasmids were analyzed by PCR to confirm the presence of insert, after which PCR product of the Cry1A(c) and Cry2Ab transgene of Bt-cotton was produced to be cloned and ligated with vector.