Aug 10, 2022

Public workspaceKasson Lab DNA Extraction

DOI
dx.doi.org/10.17504/protocols.io.q26g78413lwz/v1
  • 1West Virginia University
Brian Lovett
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.q26g78413lwz/v1
Protocol Citation: Angie Macias, Matthew T Kasson, Brian Lovett 2022. Kasson Lab DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g78413lwz/v1
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: October 28, 2020
Last Modified: August 10, 2022
Protocol Integer ID: 43955
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
Reagent isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
ReagentNuclei Lysis Solution, 1000mlPromegaCatalog #A7943
ReagentCell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933
Reagentisopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
ReagentElution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558
ReagentEthyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
ReagentCell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933
ReagentProtein Precipitation Solution 350mlPromegaCatalog #A7953
Reagentisopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907
ReagentEthyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
ReagentElution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558
ReagentElution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558
Before you begin
Before you begin
Turn on hot water bath, set to Temperature65 °C .

Pull two Eppendorf Amount1.5 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 Reagent isopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907 .
Sketch of "I"-labeled tubes (Angie Macias).

Add Amount600 µL of ReagentCell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933 (or ReagentNuclei Lysis Solution, 1000mlPromegaCatalog #A7943() to tubes without "I".

Add Amount600 µL of Reagentisopropyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W292907 to tubes labeled with "I".

Place tube with ReagentElution buffer pH 8.0 (250 mL)Alfa AesarCatalog #J61558 into Temperature65 °C water bath.

Extraction Protocol
Extraction Protocol
1h 10m 3s
1h 10m 3s
Sterilize some metal scrapers with flame and Concentration95 % (v/v) ReagentEthyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023 .

Add 1/2 pea-sized amount of fungal tissue (young hyphae) to each tube containing ReagentCell Lysis Solution, 1000ml (for Wizard Genomic)PromegaCatalog #A7933 .

Flame-sterilize and cool scrapers between samples.
Macerate each sample with a new, sterile micropestle until tissue is homogenous.
Add tubes to a floating rack to allow samples to incubate directly in Temperature65 °C water bath for Duration00:30:00 .

30m
Remove samples and vortex for Duration00:00:03 before returning to Temperature65 °C water bath for Duration00:30:00 .

30m 3s
Place a sufficient aliquot of ReagentElution 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 Duration00:05:00 .

5m
Add Amount200 µL of ReagentProtein Precipitation Solution 350mlPromegaCatalog #A7953 to each tube and vortex for 10 seconds.

Centrifuge samples for Duration00:03:00 at Centrifigation14.000 rpm .

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

3m
Using a P1000 micropipette, transfer supernatant to each tube containing Reagentisopropyl 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 Duration00:01:00 at Centrifigation14.000 rpm .

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

Add Amount600 µL of Concentration70 % (v/v) ReagentEthyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023 to each tube and mix gently by inversion.

Centrifuge for Duration00:01:00 at Centrifigation14.000 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 Amount100 µL of warmed ReagentElution 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 Temperature-20 °C freezer.