PLASMID DNA EXTRACTION By ALKALINE METHOD WITH VARIATIONS
@dr.muhammadimran5501
A - The yield of plasmid DNA is dependent mainly on the copy number of plasmids. For high copy number plasmid, 1.5 ml culture is sufficient to get good yield of plasmid DNA. However, more cultures required for good yield of low-copy number plasmid.
B - Firstly, the bacteria are pelleted and resuspended in a resuspension buffer. This buffer is often a basic pH, Tris buffer, which helps to denature DNA, and the metal ion chelator, EDTA
ethylenediaminetetraacetic acid) stabilizes the cell membrane by binding the divalent cations of Mg2+ and Ca2+ and inhibits DNases (enzymes that degrade DNA).
C - Next, the bacteria are lysed with strong alkali (Sodium Hydroxide (NaOH)) and detergent (Sodium Dodecy Sulfate (SDS). The SDS detergent solubilizes the phospholipids and proteins of the cell membrane resulting in cell lysis and the release of the cells contents, including the DNA, into the solution. The high concentration of sodium hydroxide denatures the genomic and plasmid DNA, as well ass cellular proteins. The cellular DNA becomes linearized and the strands are separated, whereas the plasmid DNA is circular and remains topologically constrained (the two strands, although denatured remain together).
D - Finally, a neutralization buffer of potassium or sodium acetate is added to neutralize the strong alkaline conditions causing the precipitation of an SDS-protein complex as a white precipitate, consisting of SDS, lipids and proteins. In addition, the potassium acetate neutralizes the solution following the renaturation of the DNA. Since plasmid DNA is covalently closed, it reanneals while genomic DNA form precipitate so the large chromosomal DNA is captured in the precipitate, whereas the small plasmid DNA remains in solution. The precipitate and chromosomal DNA is removed by centrifugation
Following centrifugation, the soluble plasmid DNA can be purified from the solution by various techniques. The most common is to precipitate the DNA with alcohol (ethanol or isopropanol).
ips:
Bacterial pelleting and Resuspension STEP
To remove the medium completely, decant the medium from the Eppendorf tube after
centrifugation. Invert Eppendorf tube upside down on a paper towel to remove residual liquid.
Gently tap the tube on the paper towel to remove liquid sticking on the sides of the tube.
To take more bacterial culture (more than 1.5 ml) for plasmid isolation, repeat the process by
adding more culture in the same Eppendorf tube. Microcentrifuge tube with 2 ml capacity can
also be used.