Category: Molecular Cloning

In contrast to conventional cloning strategies, Restriction enzyme-free cloning (also called Restriction-free cloning) involves no treatment of DNA fragments to…

ATP-dependent ligase enzyme from Bacteriophage T4 Catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl…

TA cloning involves base pairing of adenine (A) and thymine (T)  (complementary base pairs) that result in the joining of…

Molecular cloning or DNA cloning refers to a set of methods that involve in generating identical copies of a piece…

Alkaline lysis method of plasmid isolation was originally developed by Birnboim & Doly (1979). In this procedure, bacteria harbouring the desired…

To isolate plasmid from the host bacteria, cells are first lysed that lead to the release of plasmid and in subsequent steps, the plasmid is purified from the lysate. Purification of plasmid from the lysed cells are mostly dependent on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupts the bacterial cells leading to the release of cell components including both plasmid DNA and genomic DNA in denatured state relies on selective renaturation of only plasmid DNA in a perfect manner at the purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells. The purified plasmid can be further purified by a number of methods to obtain high quality plasmids. These methods are centrifugation in gradients of CsCl – ethidium bromide (EtBr), selective precipitation in high salt SDS, extraction with Phenol-chloroform, and hydroxylapatite and ion-exchange chromatography.