On cooling, five hundred microliter (500 µl) of chloroform was
added again to the mixture and mixed for 5 min. The resultant
mixture was then centrifuged at 10,000 rpm for 10 min in order to
separate each component in the homogenous mixture. The
supernatant was collected into a fresh Eppendorf tube without
taking the white phase. Five hundred microliter of both cold
isopropanol and absolute ethanol were added to the supernatant.
The mixture was kept at the temperature of -20°C inside the
refrigerator for 1 h. It was later brought out for centrifugation at
10,000 rpm for 10 min in order to separate the mixture. Afterwards,
the supernatant was decanted carefully. 500 µl of 70% ethanol was
added to the decanted supernatant which was mixed very well to
achieve a homogenous solution. This was centrifuged at 10,000
rpm for 10 min and the supernatant was discarded. The pellet left
was air dried for 1 h and then re-suspended in 200 µl sterile water.
This procedure was repeated for each of the eight mushroom
samples for 2 days. The samples were kept in sterilized PCR tubes
for subsequent DNA sequencing analysis.