Dec 15, 2021
Fungal DNA extraction for Nanopore sequencing
- Eri Ogiso-Tanaka1,
- Hiyori Itagaki2,
- Muneyuki Ohmae3,
- Tsuyoshi hosoya4,
- Kentaro Hosaka4
- 1Center for Molecular Biodiversity Research, National Museum of Nature and Science, 4-1-1, Amakubo, Tsukuba, Ibaraki, 305-0005, Japan;
- 2Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan;
- 3Hokken Co. Ltd., 7-3 Ekihigashimachi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0222, Japan;
- 4Department of Botany, National Museum of Nature and Science, 4-1-1, Amakubo, Tsukuba, Ibaraki, 305-0005, Japan
Protocol Citation: Eri Ogiso-Tanaka, Hiyori Itagaki, Muneyuki Ohmae, Tsuyoshi hosoya, Kentaro Hosaka 2021. Fungal DNA extraction for Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.b2vfqe3n
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: December 15, 2021
Last Modified: July 26, 2022
Protocol Integer ID: 55943
Keywords: fungi, DNA extraction, HMW, long read sequencing, Nanopore, National Museum of Nature and Science
Abstract
This protocol is intended for extraction of high molecular weight DNA from fungal samples.
Before start
Cut the tip of the pipette man tips in advance to make the outlet wider.
The frozen mycelium samples are ground to fine powders using a mortar and pestle in liquid nitrogen to avoid raising the temperature of the samples for high molecular weight DNA extraction.
Add 20 mL of pre-warmed (60 °C ) lysis buffer with 800 µL ProteinaseK (FUJIFILM Wako Pure Chemical Co., Ltd. Japan) and 50 µL RNase A (Nippon Gene Material Co., Ltd. Japan) in a glass beaker.
20 mL lysis buffer [2% CTAB, 100mM TrisHCl, 20mM EDTA, 1.4M NaCl, 1% PVP)]
800 µL ProteinaseK
50 µL RNase A
60 °C
The mixture was incubated at 55 °C in a water bath with shaking for 5 min.
55 °C
00:05:00 .
5m
Add 1 vol phenol/chloroform/isoamylic alcohol (PCI) 25:24:1 and mix by inversion.
Centrifuge 12000 rpm (13000 g) with soft/slow break function for 20 min at RT.
12000 rpm 13000 x g
00:20:00
Room temperature
20m
Remove aqueous layer to new 50 mL tube.
Repeat step 4
Add 1 vol chloroform and mix by inversion.
Safety information
Always work with chloroform in a fume hood.
Centrifuge 14000 rpm (17800 g) with soft/slow break function for 10 min at RT.
14000 rpm 17800 x g
00:10:00
Room temperature
10m
Remove aqueous layer to new 50 mL tube.
Repeat step 5
Add 1 vol isopropanol and mix gently by inversion.
Centrifuge 14000 rpm for 5 min at 4 °C. Remove and discard the supernatant.
14000 rpm 17800 x g
00:05:00
4 °C
5m
Rinse pellet with 1mL 75% ethanol at RT.
1 mL 75% ethanol
All aqueous and pellet to new 2 mL tube.
Spin down and remove supernatant.
Air dry DNA pellet.
Note
If you have a good nose, you can use the smell to determine if the ethanol is gone.
Resuspend DNA in sterile 50uL 10mM Tris-HCl for 1 hr~ at RT in dark.
50 µL Tris-HCl
Room temperature
01:00:00 ~Overnight
Note
DO NOT vortex or pipet to resuspend. Gently flick the tube and leave room temperature in dark for up to overnight.
2h
Check the quality and concentration of DNA using spectrophotometrically with NanoDrop (Thermo Fisher Scientific, USA) and in a Qubit 2.0 fluorometer (Thermo Fisher Scientific).
Check the DNA degradation using Genomic DNA ScreenTape assay with 2200 TapeStation system (Agilent Technologies, Germany).
Store at 4 °C until library preparation. (at -20 °C for long-term storage)