All things fungi
Camille Truong, July 2022.
In our experience, this protocol works better than many expensive kits to extract high quality DNA from a variety of fungal sources (fresh and dry specimens, fungarium, cultures, mycorrhizal roots) including for genome sequencing.
Back to mycology protocols
10 g CTAB solid
288 ml deionized water
50 ml Tris 1M
140 ml NaCl 5M
20 ml EDTA 0.5M
Sterilize by autoclaving for 20 min at 120ºC
In the field, sterilize a razor blade with a flame and/or an alcohol wipe and use it to collect fresh material (gills, gleba, root tip) in an Eppendorf tube with 500 µl 2% CTAB extraction buffer; store at –20ºC before DNA extraction.
CTAB keeps well at room temperature for several weeks/months
Using fresh material is ideal but it works for dried material too
Back to the lab, break down material inside the tube with a sterile pestle while incubating at 65ºC for >30 min; vortex sample every 10 min.
Optional: 2 µl β-mercaptoethanol can be added at this step to reduce polyphenol contaminations in genomic DNA
Add 500 µl isoamyl-chloroform [1:24]; vortex and centrifuge at max speed (>13,000 rpm) for 20 min.
Transfer the top phase to a fresh tube (ca. 300-400 µl, using yellow pipet tip) without disturbing the interface.
At this stage, the samples can be left in the freezer overnight
Centrifuge at max speed for 15 min; pour out isopropanol, being careful not to loose the pellet (use pipet tip if necessary).
Wash the pellet with 500µl 70% ethanol; mix gently and centrifuge at 12,000 rpm for 30 sec. Discard supernatant. Repeat.
Pour out ethanol (by hand), being careful to not loose the pellet; completely dry the pellet at 65ºC (>30 min) in a dry heater or vacuum centrifuge.
Resuspend DNA by adding 50-100 µl TE buffer (pH 8) or sterile deionized water; incubate at 65ºC for >20 min to degrade potential DNAse residues.
Optional: once DNA is resuspended, add 1 µl RNAse (final concentration 20 µg/ml) and incubate for >20 min