Dec 08, 2023

Public workspaceHMW DNA extraction protocol for ferns

DOI
dx.doi.org/10.17504/protocols.io.bp2l69kb1lqe/v1
  • 1Núcleo de Estudos da Vegetação Antártica - NEVA;
  • 2Universidade Federal do Pampa
Rafael Plá Matielo Lemos
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l69kb1lqe/v1
Protocol Citation: Geferson Fernando Metz, Rafael Plá Matielo Lemos, Cristiane Barbosa D'Oliveira Matielo, Tiego Ferreira, Filipe Victoria 2023. HMW DNA extraction protocol for ferns. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69kb1lqe/v1
Manuscript citation:
Geferson Fernando Metz, Tiego Ferreira, Cristiane Barbosa D'Oliveira Matielo, Rafael Plá Matielo Lemos, Filipe de Carvalho Victoria. HMW DNA extraction protocol for ferns. protocols.io
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 25, 2022
Last Modified: December 08, 2023
Protocol Integer ID: 69196
Keywords: Ferns, Nanopore, HMW, long reads, Plant DNA
Funders Acknowledgement:
Auxílio a Pesquisa GENO-ISLAND: Adaptações moleculares das plantas aos ambientes insulares
Grant ID: CNPQ 443237/2019-0
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
Grant ID: CAPES 001
Abstract
Among the difficulties encountered in a laboratory, simple situations such as efficiently disinfecting fern spores and extracting large amounts of DNA with low tissue input in the protocol are one of the major impediments in carrying out sequencing of these plants in the axenic state. The objective of this work is to present a replicable, scalable and easy-to-execute protocol for work with ferns following the current generation of long-read sequencing. Our method is based on providing a disinfection protocol for spores and sporangia that guarantees growth free of contamination and, after this growth, DNA extraction using a low amount of material in order to obtain a good yield. The results are promising since, in up to 21 days, we obtained germinated plants. After their growth (in average 180 days) we were able to extract DNA in quantity and quality and perform the sequencing, emphasizing that our best N50 is 24 Kb.
Image Attribution
Illustration by Crix D'Oliveira.
Materials
ReagentLiquid nitrogen
ReagentChloroform: Isoamyl Alcohol (24:1)
ReagentIsopropanol
ReagentTE Buffer
Reagent3M sodium acetate
ReagentEthanol 100%
Reagentnuclease free water
ReagentSodium Hypochlorite Solution
Reagent70% Ethanol
β-mercaptoethanol
PVP 40
TRIS 1 M pH 8.0
EDTA 0.5 M pH 8
Sodium Metabisulfite
NaCl 2.5M
Sodium dodecyl sulfate (SDS) 20%
2 ml LoBind tubes
1.5 ml LoBind tubes
Mortar & pestle
Water Bath
Centrifuge
Protocol materials
ReagentChloroform: Isoamyl Alcohol (24:1)
Materials, Step 15
Reagentnuclease free water
Materials, Step 26
Reagent70% Ethanol
In Materials and 2 steps
ReagentSodium Hypochlorite Solution
Materials, Step 6
ReagentLiquid nitrogen
Materials, Step 10
ReagentTE Buffer
Materials, Step 20
ReagentEthanol 100%
Materials, Step 21
ReagentIsopropanol
Materials, Step 18
Reagent3M sodium acetate
Materials, Step 20
Safety warnings
Attention

Safety information
Work under fume hood when add β-mercaptoethanol and Chloroform.


Safety information
Be careful when handling liquid nitrogen.


Before start
  • Prepare the SDS Lysis Buffer on the day of the experiment;

ABC
ReagentReagent Stock ConcentrationFINAL
PVP 40100%1%
Sodium Metabisulfite1%1%
NaCl5 M0,5 M
TRIS HCL pH 81 M100 mM
EDTA pH 80.5 M50 mM
DDH2O-~
Sodium dodecyl sulfate (SDS)20%1,5%
β-MERCAPTOETANOL-2% (v/v)
  • When preparing the SDS Lysis Buffer, add SDS at last this will avoiding bubble formation.

  • Preheat the water bath; Keep the SDS Lysis Buffer at 65°C until the tissue powder is added.

  • Washing solution (EtOH 70%), fresh
35 ml Ethanol 100% + 15 ml H2O

  • Potassium Acetate 5M
Dissolve 4.9 gr KAc in 10 ml ddH2O (4.9 gr KAc + ≈ 7.5 ml H2O).
Adjust the pH with glacial acetic acid.

  • Prepare TE buffer (10 mM Tris pH 8 and 1 mM EDTA pH 8)
Plant material sterilization
Plant material sterilization
3d 0h 45m
Figure 1. Step by step plant spore and sporangia sterilization. Illustration by Crix D'Oliveira.

Sample and store the leaf tissue (fronds) in paper envelopes for three days to induce dehiscence.
Recover and store the spores together with sporangia in 1.5 mL microtubes until one-third of the tube was filled and then stored at Temperature-20 °C until disinfection.

Add Amount1 mL Reagent70% Ethanol Contributed by users , homogenize by inversion for Duration00:00:20 and briefly centrifuge them.

20s
Discard the supernatant and wash by inversion with Amount1 mL of autoclaved purified water, briefly centrifuge and discard the supernatant.

2m
Add Amount1 mL of an ReagentSodium Hypochlorite Solution Contributed by users (active chlorine ~2%) at Concentration10 % (v/v) , homogenize by inversion for Duration00:20:00 , centrifuge at Centrifigation5.000 rpm Duration00:03:00 .

23m
Discard the supernatant and wash by inversion with Amount1 mL of autoclaved purified water, centrifuge at 5Centrifigation5.000 rpm Duration00:03:00 . Repeat this step 4 times.

3m
Add Amount1 mL autoclaved purified water, homogenize and pipete into a Petri Dish with BCD medium (MgS04.7H2O - 0.1 mM, KH2PO4 - 1.84 mM, KNO3 - 1M, FeSO4.7H2O - 4.5mM).
1m
Extraction of high-molecular-weight DNA
Extraction of high-molecular-weight DNA
2h 31m
Figure 2. Step by step extraction of high-molecular-weight DNA illustrated protocol. Illustration by Crix D'Oliveira.
Weigh between Amount50 mg and Amount100 mg of leaf tissue. Grind must be done with a crucible and pestle (previously exposed to ReagentLiquid nitrogenContributed by users ) until a very fine powder is obtained.

7m
Pre-heat the Lysis Buffer and aliquot Amount600 µL for each sample individually in 1.5 mL microtube. With the aid of a spoon or spatula, transfer the macerate to the microtube.

1m
Homogenize by inversion 10 times and incubate at Temperature60 °C for Duration00:10:00 .

10m
Add Amount4 µL of RNase A (100 mg/mL); add Amount4 µL of proteinase K (> 40 U/mg);

Homogenize by inversion 10 times and incubate at Temperature60 °C for Duration00:20:00 gently homogenizing by inversion every Duration00:05:00 .

25m
Add Amount600 µL of ReagentChloroform: Isoamyl Alcohol (24:1)Contributed by users and homogenize by inversion for at least Duration00:03:00 until an off-white emulsion forms.

3m
Centrifuge at Centrifigation14.000 rpm for Duration00:05:00 at room temperature.

5m
Carefully aspirate the upper phase of the tube and transfer to a new 1.5 mL microtube.
1m
Add Amount400 µL of ReagentIsopropanolContributed by users Temperature-20 °C and incubate it for at least Duration01:00:00 at Temperature-20 °C . (Can be stored overnight)

1h
Centrifuge at Centrifigation14.000 rpm for Duration00:05:00 at room temperature and discard the supernatant.

5m
Add Amount50 µL of ReagentTE Buffer Contributed by users . Add Amount5 µL of Reagent3M sodium acetateContributed by users .

1m
Add Amount120 µL of iced ReagentEthanol 100%Contributed by users (Temperature-20 °C ) and mix well by flicking the tube.

Store tubes at Temperature-20 °C for at least Duration00:20:00 .

20m
Centrifuge at Centrifigation14.000 rpm for Duration00:15:00 at room temperature and carefully discard the supernatant.

15m
Add Amount1 mL of freshly prepared Reagent70% Ethanol Contributed by users , mix gently by inversion, centrifuge briefly and carefully discard the supernatant.

1m
Amount50 µL Dry the pellet for approximately Duration00:10:00 at Room Temperature on the bench with the tube upside down.

10m
Elute in Amount50 µL volume in Reagentnuclease free waterContributed by users . (Elution volume can be adjusted for your needs)

1m
Quality Assessment
Quality Assessment
Quantify the DNA on a Qubit® fluorometer (dsDNA high sensitivity assay). DNA yield can be 500 - 1500 ng.
Check DNA integrity though electrophoresis in a 1'% Agarose Gel.
DNA Size Selection
DNA Size Selection
Remove short DNA fragments with Circulomics® Short-Read Eliminator Kit.