Rapid DNA extraction for fungi

Camille Truong, July 2022 (original protocol from Vilgalys Lab, Duke University)

This protocol is aimed at getting DNA from fresh sporocarps, ectomycorrhizal roots or cultures for DNA barcoding (SSU, ITS, LSU). Fungarium specimens, complex environmental samples or single-copy gene sequencing will work better with CTAB.

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Extraction solution ES (100 ml):

10 ml 1M Tris (pH 8)

1.86 g KCL

0.37 g EDTA

80 ml deionized water

• Shake until the solutes disolve
• Titrate to pH 9.5-10 with 1M NaOH
• Top up with deionized water to 100 ml
• Filter sterilize into Eppendorf tubes

Dilution solution DS (100 ml):

• Disolve 3 g BSA (>98%, heat shock fractionated) in a clean vessel
• Top up with deionized water to 100 ml
• Filter sterilize into Eppendorf tubes

Procedure

• Prepare up to 100 µl of ES into PCR tubes.

• Sample tissue with a sterilized blade or forceps and submerge into ES solution; store at –20ºC before DNA extraction.

  Avoid stuffing too much material into the tube, less is more here

  Be sure not to add any extra water or liquid (if picking root tips, blot dry the forceps)

• On the day of extraction, smash the material inside the tube using sterile forcepcs.
• Incubate for 10 min at room temperature, then 10 min at 95ºC.
• Add an equal voulme of DS to the tube so that the ratio ES:DS is 1:1

• Store at –20ºC

  Samples are now ready for PCR; 1-2 µl DNA per reaction is usually sufficent