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extracted with IS ml of urtraction buffer (T'ris-HCI pH 8 0, 20 mM EDTA, 1 4 M NaCl
an3 2.5% (wlv) CTAB) The contents were taken in 30 ml centrifuge tubes and incubated
in a water bath for 5 min at 65 C along with 0 03% fl Mercaptoethanol The tubes were
incubated in a water bath at 65 C for 30 min samples are mixed for every 5 rnin Fifteen
ml of chloroform isoarnyl alcohol (24 1) was added to each tube, mixed and centrifuged
at 6000 rpm for 10 min Top layer was separated with filler and 0 7 vol of cold
isopropanol was added and incubated at 4 C for 30 min prior to centrifugation at 6000
rpm for 15 min The pellet was transferred to 2 ml eppendorfs and washed with 2 ml of
70% ethanol, after discarding ethanol pellets were airdried before suspending in Irnl of
TE butier (50 mM Tris-HCI pH 8 0, IOmM EDTA) and DNA was transferred into fresh
eppendorfs containing 150 tJ of 50 mg/d Rnase The tubes were incubated overnight at
37 C 1.5 ml of phenol:chloroform isoarnyl alcohol in 25 24 1 ratio was added and kept
for centrihgation for 5 min at 14,000 rpm Afier thorough mixing the top layer was
separated and collected into fresh eppendorfs and 600 pl of ice-cold chloroform isoamyl
alcohol (24.1) was added. The tubes were kept for centrifugation at 14,000 rpm for 5min
and the top layer was separated again and 1/10' vol of sodium acetate (50 4 I500 4)
and 2 vol. of absolute ethanol were added, mixed gently and centrifuged at 14.000 rpm
for 5min at the room ternpzrature
The DNA was washed twice with 250 pl of 70% c thanol The pellet was fandried for
Smin and suspended in varying volumes of T E (I OmM Tris-HCI, pH 8 0.0 ImM EDTA)
buffer depending on pellet size The DNA was quantified using two methods