2.4. Method 1
Initially, in method 1, the physical force in the form of sonication is applied to the sample (kept in falcon tubes placed in ice) to rupture the cell wall at medium amplitude with three cycles per sample. The sonicated samples are transferred to a new microcentrifuge tube and spiked with 800 µl of 2% CTAB isolation buffer, followed by three cycles of snap-chill-thaw, and added 800 µl of Chloroform: isoamyl alcohol (24:1) and finally with 600 µl Phenol: Chloroform: isoamyl alcohol (25:24:1) with supernatant discarded and pelleted by centrifugation for each of the above reagent steps. The pelleted DNA was subsequently washed with 1 ml of 70% (70%EtOH+30% Nuclease free water V/V) ice-cold ethanol and re-suspended in 30-40 µl of TE buffer (pH 8) and quality/quantity checked using Quantus fluorimeter method (Cat# E6150 Promega Technologies, Madison, USA).