Preparation of Plasmid DNA

Preparation of Plasmid DNA: A culture of cell containing plasmid grown in liquid medium is harvested and cell extract is prepared. Separation of plasmid from bacterial DNA is important. Plasmid is different from bacterial DNA in size and conformation. Both plasmid and host chromosome are circular but during preparation of extract the bacterial chromosome is always broken down into linear fragments. hence, can be separated on basis of linear or circular. Separation on basis of size: Lysed cell DNA are comparatively larger than plasmid DNA hence can be removed with cell debris by cetrifugation because bacterial chromosome is attached with cell envelope. Lysis is done carefully in presence of sucrose, treated with EDTA and lysozyme.Sucrose prevent from immediate cell burst. Instead sphearoplast are formed, which is cell with intact cytoplasm membrane and partialy degraded cell wall. To it,now ionic detergent TRiton-X100 is added for cell lysis. Ionic detergent cause chromosome breakage. Then centrifugation is done which leaves clear cell lysate. Lysate contain almost entire plasmid. Separation on basis of conformation: There are two types of DNA 1. Supercoiled or covalently closed circular DNA: During plasmid replication double helix of DNA is partially unwound with the help of topoisomerase, hence take the supercoiled conformation. This conformation is maintained if both strands are intact. 2. Open circular: If one of the polynucleotide strand is broken the double helix revert to relaxed state and plasmid takes open circular state. Supercoiled are easily seperated.