Fungal DNA Isolation

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

Procedure

1. Place 0.2–0.5 g (dry weight) lyophilized pad in a 50-mL disposable centrifuge tube, break up the pad with a spatula or glass rod, add C. carbonum 5 mL 3 mm 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 of the powdered pad.
3. Place in a 65°C water bath for 30 min.
4. Cool, add equal volume of CHCl₂:IAA (24:1).
5. Mix, centrifuge in a table top fuge 10 min at full speed.
6. Transfer aqueous supernatant to a new tube.
7. Add an equal volume of isopropanol.
8. 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, and add 1–5 mL TE containing 20 µg/mL RNAse A. To resuspend the samples, place in a 65°C bath, or allow pellets to resuspend overnight at 4°C.

Notes
If the spooled DNA is discolored or has contaminating mycelial debris, phenol/chloroform extract and precipitate with ethanol.

This protocol can be scaled down using a 0.1-g pad in a 2-mL eppendorf tube.

For Southerns, we routinely cut 50–75 µL (2–4 µg) of standard DNA, prepare µL volumes, EtOH precipitate and resuspend in 30 µL.

Even after digestion the resuspended DNA can be very viscous at room temperature. To load a Southern, we keep the samples in a 50°C–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), 0.7 M NaCl, 10 mM EDTA, 1% 2-mercaptoethanol. Add proteinase K to a final concentration of 0.3 mg/mL prior to use. Less proteinase K may be acceptable for different fungi, and it hasn’t been determined if less can be used. This concentration was calibrated for a different carbonumDNA extraction buffer.

Mini Plasmid Preparation

1. Grow overnight in 1.5 mL LMM or Terrific broth (see Reagents) with 75 µg/ mL Amp.
2. Pour it into an eppendorf tube and spin down cells at 7–8 K for 2 min.
3. Aspirate and resuspend in 50 µL 25 mM Tris pH 8, 10 mM EDTA; leave lids open.
4. Add 100 µL of freshly prepared 1% SDS, 0.2M NaOH (5 mL = 100 µL 10M NaOH added to 4.4 mL DDW, then 500 µL 10% SDS). Add it forcefully and you don’t need to vortex.
5. Add 75 µL KOAc solution and vortex.
6. Add 100 µL of phenol/CHI₃, close lids, vortex.
7. Spin 13K for 2 mins.
8. Remove supernatant, add to 500 µL ethanol. Vortex and spin at 13 K for 5 min.
9. Aspirate, removing all ethanol.
10. Resuspend in 50 µL TE.
11. Digest 2–5 µL, adding 1 µL preboiled 10 mg/mL RNAse A (see Reagents for preparation).
    KOAc solution:
    60 mL 5M potassium acetate
    11.5 mL glacial acetic acid
    28.5 mL DDW
    
    Large-scale plasmid preps can be made quickly by scaling all reagents of the “mini-prep” procedure 1000-fold. Once the phenol step has been completed, treat the supernatant with 50 mL 10 mg/mL RNAse for 1 hr, isopropanol precipitate (0.6 volumes) and resuspend DNA in 400 mL of TE, reprecipitate. If the precipitate is very cloudy (residual RNA), hook the DNA out (fluffy ball) to separate it from the RNA.

Large-Scale Plasmid Preps

1. Prepare an overnight culture of 100–200 mL in Terrific broth with 75 µg/mL ampicillin.
2. Spin down cells 5K, 5 min.
3. Resuspend in 5 mL of 25 mM Tris, 10 mM EDTA pH 8.0 per 200 mL.
4. Add 10 mL of freshly prepared 1% SDS, 0.2M NaOH (50 mL = 1 mL 10M NaOH added to 44 mL DDW then 5 mL 10% SDS). Swirl to lyse cells completely and then add 7.5 mL KOAc solution per 200 mL. Spin 7 K for 10 mins.
5. Pour supernatant through cheesecloth into an SS34 tube and add 11 mL of isopropanol (for a 200-mL prep).
6. Spin 7K for 10 min. Drain off all supernatant, being careful to wipe around the inside neck of the bottle to remove all the isopropanol.
7. Resuspend pellet in 2.4 mL TE. When dissolved thoroughly, add 4.4 g CsCl. Dissolve first (handy hint) then add 40 µL 10 mg/mL EtBr. Make sure all CsCl is in the solution.
8. Layer plasmid solution under 8.5 mL of CsCl/TE pl. 47 in a Ti70 tube and spin overnight at 50K. CsCl/TE [1.47] = Dissolve 76.68 gms of CsCl in every 100 mL TE.
9. Pull band in 1.0 mL or less. Extract 3 times against butanol saturated in TE (first extraction into ethidium waste, remainder into butanol waste).
10. Add 1.0 mL DDW, estimate volume and add 2.5 vol. ethanol (no salt). Do not chill. Spin at 3K in the benchtop for 10 min (provided there was a good band to begin with, otherwise in the ultracentrifuge for 20 minutes at 20K). Pour off and remove residue with a gilson, resuspend in 350 µL TE. Read OD 260/280 on 5 µL and reprecipitate remainder in ependorf with 35 µL 5M NaCl and 1.0 mL ethanol. Resuspend at 1 µg/L.