John M. S. Bartlett.pdf - Bio-Nica.info

54 Stirling
3. Methods
3.1. Fungal Protocol
1. Grind 0.2 to 0.5 g (dry weight) of lyophilized mycellar pad in a mortar and pestle. Transfer
to a 50-mL disposable centrifuge tube.
2. Add 10 mL (for a 0.5 g pad) of CTAB extraction buffer.
3. Gently mix to wet all the powdered pad.
4. Place in 65°C water bath for 30 min.
5. Cool and add an equal volume of chloroform/isoamyl alcohol (241).
6. Mix and centrifuge at 2000g for 10 min at room temperature.
7. Transfer aqueous supernatant to a new tube.
8. Add an equal volume of isopropanol.
9. High molecular weight DNA should precipitate upon mixing and can be spooled out with
a glass rod or hook.
10. Rinse the spooled DNA with 70% ethanol.
11. Air dry, add 1 to 5 mL of TE containing 20 µg/ mL RNAse A. To resuspend the samples,
place in 65°C bath or allow pellets to resuspend overnight at 4°C.
3.2. Yeast Protocol
1. Collect cells from fresh 5 mL culture by centrifugation at 2000g for 10 min and resuspend
in 0.5 mL of water.
2. Transfer cells to 1.5-mL microfuge tube and collect by centrifugation at 15,000g for 10 min,
pour off supernatant and resuspend in residual liquid.
3. Add 0.2 mL of buffer A, 200 µL of glass beads, and 0.2 mL of phenolchloroformisoamyl
alcohol (25241).
4. Vortex for 3 min and add 0.2 mL of TE.
5. Centrifuge at 15,000g for 5 min and then transfer aqueous to new tube.
6. Add 1 mL of 100% EtOH (room temperature), invert tube to mix, and centrifuge at
15,000g for 2 min.
7. Discard supernatant and resuspend pellet in 0.4 mL of TE (no need to dry pellet).
8. Add 10 µL of 4 M ammonium acetate, mix, and then add 1 mL of 100% EtOH and mix.
9. Centrifuge at 15,000g for 2 min and dry pellet. Resuspend in 50 µL of TE.
3.3. Bacterial DNA Protocol
1. Collect the bacteria from a 15-mL overnight culture into a 1.5-mL microfuge tube.
2. Resuspend pellet with 300 µL of STET buffer and add 30 µL of RNAse/lysozyme
mixture.
3. Boil for 1 min 15 s.
4. Centrifuge at 15,000g for at least 15 min.
5. Take supernatant and phenol extract with 150 µL of STET-saturated phenol.
6. Spin and take supernatant. Add 1/10 volume 4 M lithium chloride (autoclaved). Let sit
on ice for 5 to 10 min.
7. Spin and take supernatant. Add equal volume isopropanol at room temperature and
incubate for 5 min.
8. Centrifuge at 15,000g for at least 15 min. No pellet will be visible.
9. IMPORTANT: wash with 80% ethanol (95% will cause the residual Triton to precipitate).
10. Resuspend pellet in 50 to 200 µL of TE.