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).