Extraction of DNA from E coli

Demonstration of the extraction of DNA from E.coli. Cells were harvested, pelleted and diluted in TE Buffer pH 8.0 [0.15 M NaCl; 1 mM EDTA; 10 mM Tris]. 1 mL of 10 mg/mL Lysozyme was added to break down the cell wall. 2 mL of 20 % (w/v) SDS was added to dissolve the exposed plasma membrane and lyse the cell. NaCl was added to a final concentration of 1 M, to dissociated any proteins bound to the bacterial DNA. An equal volume of Chloroform: Isoamyl Alcohol (24:1) was added to precipitate the proteins. Centrifuge separated the aqueous from the organic layer, with proteins either dissolved in the organic layer, or stuck between the two layer at the interphase. The upper aqueous layer, containing the DNA, was removed, leaving behind the protein. 100% Ethanol was added to a final concentration of 67% (v/v), to precipitate the DNA out of solution. A glass Pasteur pipette was used to bind the DNA and transfer it to a new test tube. Finally the DNA was dissolved in TE Buffer.
UV Spectrophotometric analysis resulted in an absorbance of 12.81 at 260 nm wavelength & 6.63 at 280 nm wavelength. The ratio of 260:280 of 1.93 indicates the DNA sample to no be contaminated with proteins. The 12.81 at 260 indicates the DNA concentration to be 640.5 ug/mL in 5 mL TE Buffer.