Cloning of bHLH promoter fragment in pGEMT vector using E. coli (Migula) strain for various stress tolerance in chickpea

Abstract

Genomic DNA of chickpea was isolated for bHLH promoter and amplicon of size 1.7 kb was obtained by PCR of template DNA and its ligation with pGEMT vector was performed for cloning. E. coli strain DH5α was transformed with the ligated product. The colonies containing plasmids cloned with the gene of interest remains white while the other colonies turn blue. The white colonies were selected for further screening. Gene was correctly cloned in colony number 1,2, 3,4,5,6 and 8 whereas in colony number 7 and 9, the gene did not enter the plasmid. Hence, plasmid were isolated from the confirmed colonies and Plasmid PCR was performed with P_F/P_R and M13_F/P_R primer combinations and amplicon of size 1.85 kb was obtained validating the presence of insert. Digestion of pGEMT-bHLH promoter was performed with HindIII and SalI restriction enzymes to further confirm the presence of insert. After digestion, gel was run which showed two fragments, one of vector and the other corresponding to the size of insert (1.8 Kb).Thus, validating the presence of bHLH promoter in the vector.