Cryptococcus neoformans DNA Extraction Method

Grow a 50mL YPD culture overnight (16h 0m 0s), shaking at 30°C.

Pellet cells in tabletop centrifuge in a 50 ml disposable tube.

Freeze cells at -20°Cto -80°C for <30 min, then dry in a freeze drying machine.

Add the equivalent of 3mL to 5mLof 2 mm glass beads and vortex/shake until the cell pellet is broken and a fine powder is created.

In fume hood , add 10mL CTAB extraction buffer (see Guidelines) and mix.

Incubate at65°C for 0h 30m 0s.

In fume hood , add 10mL chloroform and gently mix for approximately 0h 1m 0s.

Spin in a table top centrifuge for 0h 10m 0s (2,500 – 3,000 rpm).

Remove supernatant (c. 7 ml) and add to an equal volume of isopropanol in a 15 ml disposable tube.

Gently rock back and forward to mix.

If the DNA precipitates in strands and clumps, spool out with a glass pipette and transfer to eppendorf containing 1mL70% ethanol.

Otherwise, spin in a table top centrifuge for 0h 10m 0s, pour off supernatant and use 1mL 70% ethanol to wash DNA pellet and transfer it to an eppendorf tube.

Spin sample in microcentrifuge for 5-10 minutes. Remove ethanol and allow to air dry.

Resuspend DNA in either water or TE buffer (c. 500 μl).

RNase can be added to final concentration of 20 μg/ml if needed.

Run 1µL on an agarose gel to check concentration and quality.