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1. FUNGAL DNA
ISOLATION
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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.