This protocol was developed by Kerry O’Donnell at USDA-ARS (Kerry.ODonnell@ars.usda.gov).

1 ) Add lyophylized, pulverized mycelium ~1/2 way up conical portion of 1.5 mL eppenforf tube [~50-100 mgs]– use yellow pipette tip to pulverize mycelium

2 ) Add 700 μL CTAB buffer [100 mM Tris-Cl pH 8.4, 1.4 M NaCl, 25 mM EDTA, 2% CTAB] and vortex to resuspend mycelial powder – use yellow pipette tip to resuspend all material

3 ) In hood, add 700 μL chloroform, shake back-and-forth 3-4 times

4) Centrifuge for 10 min at max speed

5 ) In hood, remove upper phase (~350 μL) to new 1.5 mL eppendorf tube
[try to remove a fixed volume for all samples]

6 ) Precipitate DNA with 350 μL (i.e., equal volume) -20°C isopropanol — invert several times — most tubes should show a visible ppt.

7 ) Centrifuge at max speed for 1-2 min

8 ) Discard soup and gently wash pellet with 70% ETOH

9) Centrifuge for 2-3 min at max speed

10) Gently discard soup, spin 10-20 sec in centrifuge to collect ETOH at bottom of tube and remove with a pipet

11) Resuspend pellet in 200 μL ddH2O or TE pH 8 – place in heat block for 10-60 min to resuspend pellet [sometimes it is necessary to draw the genomic DNA into a PCR pipet tip to help break up pellet]

12) For PCR, dilute 4 μL genomic DNA in 200 μL ddH2O in 96 well plate

Recipe for 100 mLs CTAB buffer:
10 mL 1 M Tris-Cl pH 8.4
8.18 g NaCl
5 mL 0.5 M EDTA pH 8.0
2 g CTAB [hexadecyltrimethyl-ammonium bromide; Sigma H-6269]
ddH2O to 100 mLs [dissolve CTAB by heating in ~55°C waterbath]
Recipe for Yeast-Malt Broth [per L]:
yeast extract 3 g
malt extract 3 g
peptone 5 g
glucose 20 g

Brad Cavinder in the Frances Trail lab (Department of Plant Biology at Michigan State University; trail@cns.msu.edu) developed this based on several standard protocols.

CTAB extraction buffer             100 ml                         200 ml
0.1 M Tris, pH 7.5                           10 ml                             20 ml                of 1 M stock
1% CTAB                                         1 g                                2 g
0.7 M NaCl                                       14 ml                             28 ml                of 5M stock
10 mM EDTA                                     5 ml                              10 ml                 of 0.2M stock
1% 2-mercaptoethanol                    1 ml                               2 ml
Water                                               to 100 ml                      to 200 ml

CTAB will dissolve completely only after NaCl is added, and the solution is heated to 65 ^oC and stirred. The buffer may need to be reheated occasionally.
Inoculate YES medium with one or two small bits of mycelia. Shake at 250 rpm @ room temperature for 2-3 days. Filter mycelia through miracloth, freeze and lyophilize.

Small scale extraction PCR DNA
1)  Label a 1.5 ml tube for each sample. Add a small amount of small glass beads
2)  Tare each tube and weigh out 15 mg lyophilized tissue into the tube.
3)  Close tube and place in -80 ^oC freezer for 10 min or drop tubes in liquid nitrogen for a minute.
4)  Retrieve tube; open and grind mycelia well with blue plastic pestle.
5)  Add 700 ul CTAB buffer; continue grinding. Mix well by inverting and shaking to ensure all the mycelia are suspended (turn the tube upside down and slam top on counter if needed). Do not vortex at any step.
6)  Put at 65 oC for 20 min. Invert tubes several times after the first 10 min. Cool on ice (1-2 min)
7)  Add 300 ul phenol, 300 ul chloroform:IAA (24:1). Mix well by inversion. Spin at high speed 5 min.
8 )  Transfer (upper) aqueous phase to new tube.
9)  Add 300 ul phenol, 300 ul chloroform:IAA. Mix well by inversion. Spin at high speed 5 min.
10)  Transfer aqueous phase to new tube.

11)  Add 500 ul chloroform. Mix well by inversion. Spin at high speed 5 min.

12)  Transfer aqueous phase to new tube.
13)  Add cold 100% ethanol from a -80 ^oC freezer until tube is full (800-900 ul); invert several times and place in -80 ^oC freezer for 15-30 min (until the solution has thickened but not frozen).
14)  Spin down at max speed for 10-15 min in 4 ^oC centrifuge. Pour off supernatant.
15)  Wash pellet in 600 ul ice cold 70-80% ethanol. Spin down at high speed for 1 min.
16)  Pour off supernatant. Leave pellet to dry or place on 65 ^oC heating block for 2 min.
17)  Resuspend in 50-100 ul water. (if doing >1 of the same sample, pool them here and resuspend in smallest amount of water possible)  Place on 65 ^oC heating block with open cap for several minutes to ensure all ethanol is gone. Can do the same with a closed cap to help dissolve the pellet too. Sometimes pellets resuspend better at 37 ^oC.
18)  Add 1 ul Rnase, Dnase free enzyme solution per 100 ul DNA suspension. Incubate @ 37 ^oC for at least 30 min.
19)  Add 20 ul proteinase K solution (20 mg/ml) and digest for at least 1 hour up to but not over 65 ^oC (over 65 ^oC inactivates proteinase K).
20)  Add volume 3M NaOAC then add CTAB extraction buffer to reach a volume of 700 ul, invert several times, and follow steps 7-17 above. You now have high quality DNA.

Frances Trail lab (Department of Plant Biology at Michigan State University; trail@cns.msu.edu) developed this protocol based on an article published in Mycologia (1965; vol. 67:962).

Inoculate each 250 ml Ehrlenmeyer flask containing 100 ml CMC with a few grains of soil stock or a couple of chunks of agar with mycelia.  Shake at room temp at 250rpm for about 72 hrs.

Spin down.

CMC Medium

15 g carboxymethylcellulose (Sigma-Adrich catalog no. C 5678 sodium salt, low viscosity).

1.0 g NH₄NO₃

1.0 g KH₂PO₄

0.5 g MgSO₄-7H₂O

1.0 g Yeast Extract

in 1 L H₂O

*Note: add CMC slowly or it will clump.  Use ONLY CMC stated above, others are too viscous.