真菌DNA提取

Saghai-Maroof MA, Soliman KM, Jorgensen RA, & Allard RW (1984) PNAS 81:8014-8018

DNA successfully isolated from fungal species of Cochliobolus, Aternaria, and Fusarium. The key elements in this prep are (1) the use of young lyophilized mycelial mats....young mats (4 days growth for C. carbonum)...yield less contaminating carbohydrates and other misc. junk (2) lots of proteinase K in the extraction buffer to kill DNases (final =0.3mg/ml).

Protocol

1.place 0.2- 0.5 g (dry wt) lyophilized pad in a 5o ml disposable centrifuge tube, break up the pad wtih a spatula or glass rod, add c. 5 ml 3mm glass beads and powder the pad by brief shaking.

2.add 10 ml (for a 0.5 g pad) of CTAB extraction buffer(see recipe, below), gently mix to wet all the powdered pad

3.place in 65 C water bath for 30 min

4.cool, add equal vol. ChCl3:IAA (24:1)

5.mix, centrifuge in table top fuge 10 min at full speed

6.transfer aqueous supernatant to a new tube

7.add an equal volume of isopropanol

8.High mw DNA should spool out upon mixing...spool out the DNA with a glass rod or hook, pour out the remaining supernatant

9.rinse the spooled DNA with 70% ethanol

10.air dry, add 1- 5 ml TE containing 20 ug/ ml Rnase A. To resuspend the samples, place in 65 ℃ bath, or allow pellets to resuspend overnight at 4 ℃

Notes: if the spooled DNA is discolored or has contaminating mycelial debris, phenol/chlorofrom extract and precipitate w/ ethanol.

This protocol can be scaled down using 0.1g pad in a 2ml eppendorf tube

For Southerns we routinely cut 50- 75 μl (2-4 μg) of a standard DNA prep in 200 μl rxn volumes, ETOH ppt, and resuspend in 30 μl.

Even after digestion the resuspended DNA can be very viscous at room temp; To load a Southern we keep the samples in a 50- 60 C heat block while loading to keep the samples at a lower viscosity.

CTAB extraction buffer: O.1M Tris, pH 7.5, 1% CTAB (mixed hexadecyl trimethyl ammonium bromide), 0.7M NaCl, 10mM EDTA, 1% 2-mercaptoethanol. Add proteinase K to a final concentration of 0.3 mg/ml prior to use. Less prot K may be acceptable for different fungi, and in fact we haven't determined if we can use less...this conc. was calibrated for a different C. carbonum DNA extraction buffer.