Oct 07, 2022

Public workspaceHMW DNA extraction for Long Read Sequencing using CTAB

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l694yzlqe/v1
HMW DNA extraction for Long Read Sequencing using CTAB
  • Benoît Vacherie1,
  • Karine Labadie1,
  • Cyril Falentin2
  • 1Genoscope, Institut de Biologie François Jacob, Commissariat à l'énergie atomique (CEA) Université de Paris-Saclay Evry 91057 France.;
  • 2IGEPP, INRAE, Institut Agro, Univ Rennes, Le Rheu, 35653, France.
  • HPM/Genoscope
Benoît Vacherie
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.bp2l694yzlqe/v1
Protocol Citation: Benoît Vacherie, Karine Labadie, Cyril Falentin 2022. HMW DNA extraction for Long Read Sequencing using CTAB. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l694yzlqe/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: September 15, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 70072
Keywords: extraction, high molecular weight, plant, leaves, DNA, Long read sequencing, high molecular weight gdna extraction from plant leaf, hmw dna extraction for long read sequencing, using ctab high molecular weight dna extraction protocol, hmw dna extraction, high molecular weight gdna extraction, dna extraction, conventional ctab extraction, dna fragment size selection, long read sequencing, using commercial qiagen genomic tip, extraction, commercial qiagen genomic tip, flash frozen leaf, frozen leaf, dna, plant leaf, using short read eliminator, leaf, oxford nanopore technology
Abstract
High Molecular Weight DNA extraction protocol for Long Read sequencing.
Extraction is performed from flash frozen leaves stored at -80°C.
The protocol is adapted for an extraction of 1g of leaves.
This protocol is based on the protocol provided by Oxford Nanopore Technologies, Oxford, UK (ONT), "High molecular weight gDNA extraction from plant leaves" provided by the ONT community in March 2019, with slighly modifications.
This protocol involves a conventional CTAB extraction followed by purification using commercial Qiagen Genomic tips (QIAGEN, MD, USA). DNA fragment size selection is performed using Short Read Eliminator (Circulomics, MD, USA).
This protocol is particularly adapted for plant leaves, but also works with many other organisms (microalgae, insects...).
Guidelines
Use only wide bore tips.
Work in a chemical hood when using 2-mercaptoethanol.
Allow the DNA to resuspend for at least of 24 hours before proceeding with QC.

Materials
Reagents :
ReagentTris HClP212121
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
ReagentSodium chlorideP212121
ReagentPEG-8000PromegaCatalog #V30111
ReagentLiquid nitrogen
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentRNase AQiagenCatalog #19101
ReagentChloroform
ReagentIsopropanol
ReagentGenomic tip 100GQiagenCatalog #10223
ReagentBuffer G2QiagenCatalog #1014636
ReagentBuffer QBTQiagenCatalog #19054
ReagentBuffer QCQiagenCatalog #19055
ReagentBuffer QFQiagenCatalog #19056
ReagentEthanol 70%


Consumables :

ReagentMBP™ Wide Bore Pipette TipsThermo FisherCatalog #02707600
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
Reagent15 ml conical tubes
Reagent1.5ml Eppendorf DNA LoBind tubes


Equipment :


ReagentPorcelain Mortar, 145mLThermo FisherCatalog #CP1782004
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667
ReagentVortex Mixer
ReagentGlass pasteur pipettes



Protocol materials
ReagentBuffer QFQiagenCatalog #19056
ReagentMBP™ Wide Bore Pipette TipsThermo FisherCatalog #02707600
Reagent1.5ml Eppendorf DNA LoBind tubes
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentTris HClP212121
ReagentBuffer G2QiagenCatalog #1014636
ReagentSodium chlorideP212121
ReagentLiquid nitrogen
ReagentChloroform
ReagentIsopropanol
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
ReagentGlass pasteur pipettes
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667
ReagentRNase AQiagenCatalog #19101
ReagentBuffer QBTQiagenCatalog #19054
ReagentBuffer QCQiagenCatalog #19055
ReagentEthanol 70%
Reagent15 ml conical tubes
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
ReagentPorcelain Mortar, 145mLThermo FisherCatalog #CP1782004
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentGenomic tip 100GQiagenCatalog #10223
ReagentVortex Mixer
ReagentPEG-8000PromegaCatalog #V30111
Reagent70% Ethanol
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
ReagentBuffer TE 1x
Troubleshooting
preparation of reagents
Extraction Buffer : Prepare 20 mL of extraction buffer (per 1g of leaves) in a 50 mL tube

ReagentsFinal concentration
Tris-HCl ph8100 mM
EDTA20 mM
CTAB2%
NaCl1.4. M
PEG 80001 %
H2OQSP 20 ml

DNA Extraction
3h
Add 50 µl of 2-Mercaptoethanol to 20 ml of extraction buffer and preheat to 65 °C (approx. 15 min).
Amount20 mL Extraction buffer
Amount50 µL Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
Temperature65 °C

Put a mortar in ice.
Cool the mortar and pestle with liquid nitrogen until the bubbling stops.
ReagentLiquid nitrogen

Grind 1g of frozen sample to a fine powder (approx. 2 min), without adding liquid nitrogen.

Amount1 g Duration00:02:00

Before grinding
After grinding
2m
Transfer the powder to the pre-warmed extraction buffer
Add 40 µl of RNase A (100mg/ml) and vortex the tube 5 sec.
Amount40 µL ReagentRNase AQiagenCatalog #19101
Duration00:00:05 Vortex

5s
Incubate 1h at 65°C with intermittent agitation (300 rpm every 10 min)
Duration01:00:00 65°C

ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667


1h
Let the tube cool for 5 min at RT
Duration00:05:00 RT

5m
Add 1 volume (20ml) of chloroform and vortex 2x5sec.
Amount20 mL ReagentChloroform

Centrifuge with low acceleration and deceleration
Centrifigation5500 x g, 4°C, 00:10:00 , Acc/Dec : 6/3
Before centrifugation
After centrifugation

10m
Gently transfer the aqueous phase to a new 50 ml tube.
Add 0.7 volume of isopropanol and mix gently by inversion 10 times
ReagentIsopropanol

Put the tube at -80°C for 15 minutes.
Duration00:15:00 -80°C

15m
If a DNA medusa appears after this step, recover the medusa using a Pasteur pipette (without breaking the tip of the pipette).
Wash the medusa in 3 successive baths of 70% ethanol then resuspend the medusa in 100µl of 1X TE.
Incubate for 1 hour at 55°C then store the tube at 4°C before quality control.
Continue the protocol with the rest of the tube (without the medusa).
If no DNA medusa is visible continue the protocol.
Critical
Centrifuge the tube with low acceleration and deceleration
Centrifigation5500 x g, 4°C, 00:30:00 , Acc/Dec : 6/3
30m
Carrefully remove the supernatant without resuspending the pellet.
Remove the remaining liquid by turning the tube upside down on a paper towel (make sure the pellet does not come off).
Gently resuspend with the pipette the DNA pellet with 9.5 ml of G2 buffer (QIAGEN Genomic-tip 100/G).
ReagentBuffer G2QiagenCatalog #1014636

Incubate 15 min at 50°C. The sample can be stored overnight at 4°C at this stage.
Duration00:15:00 50°C

15m
Génomic tip purification
1h
Equilibrate a QIAGEN Genomic-tip 100/G column with 4 ml of QBT buffer.
ReagentGenomic tip 100GQiagenCatalog #10223
ReagentBuffer QBTQiagenCatalog #19054

Preheat 5 ml of QF buffer to 55°C.
ReagentBuffer QFQiagenCatalog #19056

Allow all the QBT buffer to drain by gravity flow into a 50 ml tube.
Add the sample in G2 buffer (9.5ml) on the column and let it to enter the resin by gravity flow.
Wash the column twice with 7.5 ml of QC buffer.
ReagentBuffer QCQiagenCatalog #19055

Put the column on a 15 ml tube.
Elute the DNA with 5 ml of QF buffer preheated to 55°C.
ReagentBuffer QFQiagenCatalog #19056

DNA précipitation
3h
Add 0.7 volume of isopropanol to the eluate.
Mix gently by inverting 20 times.
ReagentIsopropanol

Incubate 15 minutes at RT.
Duration00:15:00 RT

15m
Centrifuge with low acceleration and deceleration
Centrifigation5500 x g, 4°C, 00:30:00 , Acc/Dec : 6/3
30m
Carefully discard the supernatant, gently resuspend the pellet with 1 ml of cold 70% ethanol.
Reagent70% Ethanol

Transfer the resuspended DNA to a 1.5 ml DNA LoBind tube.

ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021

Centrifuge with low acceleration and deceleration
Centrifigation5000 x g, 4°C, 00:10:00 , Acc/Dec : 6/3
10m
Carefully discard the supernatant.
Air dry the pellet at RT for about 10 minutes.
Duration00:10:00 RT

10m
Resuspend the pellet with 50-100 µl of 1X TE buffer.
ReagentBuffer TE 1x

Allow the DNA to resuspend for 2 hours at 55°C or ON at RT.
Duration02:00:00 55°C or DurationOvernight RT

2h
Store the DNA at 4°C.
Sample quality control
Quantify your sample with a Qubit DNA HS assay.
Check the purity of the sample with a Nanodrop (measurements of 260/280 and 260/230 absorbance ratios).
Estimate the molecular weight of the sample with a Tapestation and/or a Femto pulse and/or a Pippin Pulse.
Depending on the DNA concentrations and DNA length profiles, deplete short DNA molecules using SRE size selection kits (SRE XS, SRE or SRE XL kits).
Results
QC results obtained on different species.