Aug 24, 2023

General Fungal DNA Extraction V.2

Version 1 is forked from Kasson Lab DNA Extraction
DOI
https://dx.doi.org/10.17504/protocols.io.3byl4q7rzvo5/v2
  • 1West Virginia University
Brian Lovett
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4q7rzvo5/v2
Protocol Citation: Angie Macias, Matthew T Kasson, Brian Lovett 2023. General Fungal DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q7rzvo5/v2Version created by Brian Lovett
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 24, 2023
Last Modified: August 24, 2023
Protocol Integer ID: 86920
Keywords: general fungal dna extraction, dna from various fungi, dna extraction, extracting dna, various fungi, fungi, molecular work such as pcr amplification, extraction method, pcr amplification, pcr, dna
Abstract
This is a routine protocol for extracting DNA from various fungi. This extraction method is suitable for follow-up molecular work such as PCR amplification.
Materials
Sterile micropestles, isopropyl alcohol, ethyl alcohol, cell lysis buffer, protein precipitation buffer, elution buffer, metal scraper.
Protocol materials
isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
Cell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933
Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943
isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
Elution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558
Ethyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
Protein Precipitation Solution 350mlPromegaCatalog #A7953
Before you begin
Turn on hot water bath, set to 65 °C .

Pull two Eppendorf 1.5 mL centrifuge tubes per sample.

Label both sets of tubes with (short) sample names.
Label one tube set for each sample with an "I" for isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907 .
Sketch of "I"-labeled tubes (drawing from Angie Macias).

Add 200 µL of Cell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933 (or Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943() to tubes without "I".

Add 600 µL of isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907 to tubes labeled with "I".

Place tube with Elution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558 into 65 °C water bath.

Extraction Protocol
1h 10m 3s
Sterilize some metal scrapers with flame and 95 % (v/v) Ethyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023 .

Add 1/2 pea-sized amount of fungal tissue (young hyphae) to each tube containing Cell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933 .
Flame-sterilize and cool scrapers between samples.
Alternatively, pellet a pea-sized amount of mycelium grown in liquid culture and transfer to each tube.
Macerate each sample with a new, sterile micropestle until tissue is homogenous.
Add 400 µL of Cell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933 (to 600 µL total volume added).
Add tubes to a floating rack to allow samples to incubate directly in 65 °C water bath for 00:30:00 .

30m
Remove samples and vortex for 00:00:03 before returning to 65 °C water bath for 00:30:00 .

30m 3s
Place a sufficient aliquot of Elution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558 in water bath to warm for Step 21.

Remove samples and allow them to cool on the bench for 00:05:00 .

5m
Add 200 µL of Protein Precipitation Solution 350mlPromegaCatalog #A7953 to each tube and vortex for 10 seconds.

Centrifuge samples for 00:03:00 at 14.000 rpm .

Note
Proteins will form a large pellet: unload samples carefully into rack.

3m
Using a P1000 micropipette, transfer supernatant to each tube containing isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907 and gently mix by inversion.
Note
It's better to leave some liquid than to carry bits of the protein pellet into the next step.


Centrifuge for 00:01:00 at 14.000 rpm .

1m
Carefully pour off the supernatant into waste container.
Note
Be careful to not lose your white DNA pellet!

Add 600 µL of 70 % (v/v) Ethyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023 to each tube and mix gently by inversion.

Centrifuge for 00:01:00 at 14.000 rpm .

1m
Repeat Step 16.
Open and invert tubes onto a clean paper towel.
Note
A tube rack can be placed on the tube lids to secure inverted tubes onto the paper towel.

Add 100 µL of warmed Elution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558 to each tube.

Store fully-labeled tubes in a box (not a tube rack) in the -20 °C freezer.