[SagarikaGhosh]

A plasmid is an extra chromosomal DNA molecule quite different from the chromosomal DNA. Plasmid DNA is capable of replicating independently from the chromosomal DNA. They are double stranded and usually circular. Plasmids are known to be naturally occurring in bacteria and a few eukaryotic organisms.

Isolation of plasmid DNA is a common routine in research laboratories. The protocol involved in its extraction is called ‘mini prep’ which yields fairly clear DNA quickly and easily.Thie following protocol is based on the one given by Birnhoim and Doily in 1979 , it is modified according to the experiments’ requirements.

The varieties of chemical solutions used in the process of DNA extraction have different roles to play.

Solution I is added to maintain the pH of mixture containing the cells. SDS solution solubilises the phospholipids and proteins which are active components of cell membrane, thereby causing lysis of the cell and release of all its components. It contains RNase which acts on the RNA in the cell and denatures it.

Solution II is added for cell lysis in which NaOH is known to cause denaturation of chromosomal and plasmid DNA as well as the protein component of the cell.

Solution II is added for renaturation of plasmid DNA. For this purpose the mixture is made acidic by adding glacial acetic acid. The genomic DNA is very large so it can’t renature, hence only plasmid DNA is obtained.

Procedure:-
• Inoculate a test tube containing 100 ml of Luria Bertani media with a single isolated colony picked from LB agar plate.
• The culture is grown overnight at 37̊ C with shaking at regular intervals.
• 1.5 ml of overnight culture is centrifuged in a micro-centrifuge tube for 20 min at 4000 rpm.
• The supernatant is discarded and bacteria are resuspended in 5 ml of ice cold solution I by vortexing or pippetting up and down.
• Transfer the suspension to 50 ml ochrage tubes.
• 8 ml of solution II is added to it and mixed by shaking it up and down.
• Store at room temperature for 10 mins.
• 5 ml of solution III is added to it and thoroughly mixed. Store on ice for 30 mins.
• As soon as solution III is added, a white ppt is formed. The mixture is again centrifuged for 15 mins at 12000 rpm.
• The supernatant is then transferred to a fresh tube and 0.7 vol of isopropanol is then added to the supernatant and mixed properly.
• It is kept at room temperature for 10 mins and again centrifuged at 12000 rpm for 20 mins at 4̊ C.
• Discard the supernatant and air dry pellet.
• Add 1 ml in 100 ml of culture 1x TAE pH 8.0.
• Split into two eppendorfs. Add 250 µl phenol and 250 µl CHCl3.
• Mix by vortexing and centrifuge at 13000 rpm for 5 min.
• Take the aqueous phase and add 2 volume of chilled ethanol.
• Store at -20̊ C till required.