The document details a protocol for isolating DNA from fungal species including Cochliobolus, Alternaria, and Fusarium, emphasizing the use of young lyophilized mycelial mats to minimize contamination. Key steps include breaking up the mycelial pad, mixing with a CTAB extraction buffer, and centrifuging to separate the DNA, which is then spooled and purified. Additional notes provide instructions for addressing contamination and insights for adjusting the protocol for different fungal types.

1. FUNGAL DNA
ISOLATION
qw
M.Phool Badshah

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

3. PROCEDURE :
 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.
 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.
 Place in a 65°C water bath for 30 min.
 Cool, add equal volume of CHCl2:IAA (24:1).
 Mix, centrifuge in a table top fuge 10 min at full speed.

4. A
 Transfer aqueous supernatant to a new tube.
 Add an equal volume of isopropanol.
 Upon mixing spool out the DNA with a glass rod or
hook. Pour out the remaining supernatant.
 Rinse the spooled DNA with 70% ethanol.
 Air dry, and add 1–5 mL TE containing 20 mg/mL
RNAse A. To resuspend the samples, place in a 65°C
bath, or allow pellets to resuspend overnight at 4°C.

5. 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 mL (2–4 mg) of
standard DNA, prepare in 200-mL volumes, EtOH
precipitate and resuspend in 30 mL.

6. A
 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 C. carbonum DNA extraction buffer.