Tag: plasmid
Plasmid Isolation
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.
Growing Escherichia coli for Plasmid Isolation
Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. The fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g., DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).
