Protocol: Fungal genomic DNA extraction
Overview: The purpose of this method is to extract DNA from the fungi isolated from mesh bag burial
study, using lyophilization/tissue grinding followed by a phenol/chloroform extraction. Most DNA
extraction kits we tested yielded very little DNA, and/or the DNA extracts inhibited the DNA
polymerases used in subsequent PCR reactions. My experience with the former is that physical methods
work better than chemical methods, so we gave up on kits and cracked open fungal cells via freezedrying followed by mechanical grinding. My experience with polymerase inhibition is that this is likely
due to secondary metabolites produced by the Ascomyete fungi we are working with. Secondary
metabolites are not as abundant in non-sporulating mycelia. Therefore, we prepared tissue for DNA
extraction by inoculating fungal liquid medium with spores and incubating in the dark – conditions which
inhibited or slowed sporulation by most of our isolates. Mycelia were harvested under vacuum
filtration, flash-frozen, lyophilized, and ground. DNA was extracted from 100mg ground tissues.
Safety:
1. Phenol is toxic and causes severe skin burns. Read the MSDS, work in the hood, and wear gloves, lab
coat, and goggles! Discard phenol waste in dedicated container in fume hood.
2. Perform all fungal inoculation and harvest steps, including grinding, in a biosafety cabinet.
Materials: For 20 extractions
60
1.5L
60
2
2
1
~120
1
Petri plates (depending upon growth rate)
Potato dextrose broth or glucose minimal medium broth (see accompanying protocol)
Eppendorf tubes
Sidearm flasks, with single-hole stoppers and glass tubing
Rubber tubing
Buchner funnel
Whatman #1 filter paper to fit in Buchner funnel
P1000, P10 automatic pipettors and filter tips
Box sterile 1 mL pipet tips
Several stacks of paper towels
Liquid nitrogen
Lyophilizer
Sterile toothpicks
LETS buffer (recipe below)
phenol: CHCl3: isoamyl alcohol (25:24:1).
100% ethanol, ice-cold
70% ethanol, freshly mixed and ice-cold
Microfuge
Microfuge in cold (4oC) room
RNase
Spectrophotometer
10mM Tris buffer (pH 8) (recipe below)
Protocol:
A. Tissue preparation – spore-free mycelial generation, harvest, and lyophilization
1.
2.
Perform Steps 2-10 in biosafety cabinet.
Add ~20 mL potato dextrose broth or glucose minimal medium broth to three petri dishes per
isolate to be cultured. Choice of medium is by trial-and-error, observing which supports growth
but not sporulation.
3. Mark with strain designation, and use sterile flat toothpicks to scrape about a BB-sized clump of
spores from a plate of inoculum. Swirl the spores to distribute evenly in one plate. Repeat for the
other two plates. For non-sporulating fungi, add several small chunks of mycelium/agar to broth.
For yeasts, inoculate 5mL of broth in a culture tube. (These should be extracted using the 5 Prime
Archivepure DNA extraction kit according to manufacturer’s direction – no lyophilizing required.)
4. Incubate at 20oC for 24-72hrs, in the dark. Harvest when growth yields ~1-2 grams wet weight.
5. Set up the Buchner funnel atop a vacuum flask (with trap flask) in the biosafety cabinet. Have a
stack of paper towels in cabinet. Mark Eppendorf tubes with all strain designations in advance, and
poke/melt three holes in each Eppendorf tube cap using a hot (flamed) dissecting needle or
hypodermic needle.
6. Pour off media from all three plates into Buchner funnel lined with two Whatman #1 filters.
7. Use a flat-sided spatula to collect mycelial mat. Roll up like a cigar.
8. Place mycelia on a stack of paper towels and cover with another stack of paper towels. Press to
expel excess liquid. Repeat till dry.
9. Scrape tissue off towels, and place in marked Eppendorf tube.
10. Flash-freeze in liquid nitrogen.
11. Lyophilize for >10 hrs.
12. Autoclave all waste: Petri plates, paper towels, filters, and aspirate from filter flasks. Items that
cannot be autoclaved should be soaked for 10 min in a freshly-made 10% Clorox solution.
B.
Genomic DNA extraction
13. Take samples off lyophilizer and transfer to biosafety cabinet again. Break up lyophilized hyphae
into a fine powder using toothpicks. If >100 mg then discard extra.* Once tissue is suspended in
liquid (Step 15), you may work at the bench.
14. Mark 3 more sets of Eppendorf tubes beforehand for sample transfers.
15. Add 700uL of LETS buffer to ground, lyophilized mycelium. Mix by using the toothpick as well as
inverting the tubes several times. Incubate samples on bench for 5 min. The SDS in LETS buffer
lyses the cells.
16. Add 700uL of phenol: CHCl3: isoamyl alcohol (25:24:1). Mix by inverting 10-15 times. Incubate
samples at RT for 5 min.
* It is critical to not try and extract DNA from too much powdered mycelia. If you have 100 mg (look for
100 uL marking on Eppie tubes) powdered mycelia this will give you a reasonable yield of DNA. Trying to
extract it from more leads to DNA that will not digest and often is contaminated with nucleases.
In Cold room: Transfer on ice
17. Centrifuge 10 min at 4oC 13,000 x g. Take out of centrifuge gently to avoid disturbing 2 phases. Be
careful not to get phenol on sides of tubes, and thereby into the microfuge.
18. Transfer supernatant to a new Eppendorf tube. Add equal volume of phenol: CHCl3, isoamyl
alcohol (25:24:1). Incubate on ice for 5 min. Centrifuge for 10 min at 4oC at max speed.
19. Transfer supernatant to a new tube and add 1mL ice-cold 100% EtOH. Incubate on ice for 5 min.
Mix well and place in centrifuge with hinge facing outside of rotor. Spin for 10 min at 4oC at max
speed. This will pellet the DNA. The DNA will be on the hinge side of your tube, even if it is not
visible.
Back to room temp:
20. Remove supernatant and add 70% EtOH to wash pellet.
21. Spin for 2 min at room temperature at 13,000 x g. This will pellet the DNA.
22. Discard supernatant (be careful not to lose the pellet) and spin for 10 sec to get last of liquid to
bottom of tube. Carefully draw of remaining liquid with P10. Air dry pellet at RT for 5 minutes or
for 2 minutes in the biosafety cabinet with the fan running.
23. Resuspend the pellet with 50uL 10mM Tris buffer (pH 8).
24. Add 2uL RNase (10 mg/mL stock) and digest at 50oC for 30 min.
25. Heat inactivate DNase prior to long-term storage by heating to 65oC for 10 min.
26. Blank Nanodrop spectrophotometer with 10mM Tris and determine the concentration of each DNA
sample.
27. Mark sample tube with sample designation, date, concentration, type of DNA (gDNA in this case),
solvent (10 mM Tris), and your initials.
LETS Buffer
20 mM EDTA
0.5% SDS
10mM Tris-HCL (pH 8.0)
0.1M LiCl
1M Tris, pH 8.0
Mix 121.1 g of Tris base with 800 mL dH2O. Add 42 mL HCl. pH is temperature dependent: ~0.03 pH units
per 1oC increase. Make sure it is at RT before making final pH adjustments. Add dH2O to 1 L. Autoclave.