Molecular cloning is an essential technique to create DNA-based experimental tools for expression in bacterial or mammalian cells. Examples of such DNA constructs include a promoter element fused to a reporter gene or a cDNA sequence under the control of a ubiquitous promoter. Molecular cloning entails the preparation of the vector and insert DNAs, ligation of the insert into the vector, transformation of competent E. coli, and identification of positive clones Traditionally, molecular cloning is defined as the isolation and amplification of a specific DNA fragment. Most of these fragments are created either by digesting an existing piece of DNA with restriction enzymes or by targeting it via PCR. Short inserts of ~ 100 bp can also be commercially synthesized as complementary single-stranded oligos, which are subsequently annealed to form a double-stranded fragment. After successful isolation, the DNA of interest is ligated into a vector plasmid, a double-stranded circular piece of DNA that can be propagated in E. coli. Vectors used in the laboratory represent a smaller version of naturally occurring plasmids that include several basic features: a replication origin, a drug-resistance gene, and unique restriction sites to facilitate the insertion of DNA fragments. Often, several different restriction sites are clustered together in so-called ‘polylinker regions’ or ‘multiple cloning sites,’ making it easier to choose convenient and unique restriction enzyme combinations for a variety of inserts. You can share your ideas & comments further at : molecularbiology@scholarlymed.com molecularbiology@scholarlymed.com