Oct 06, 2022

Public workspaceHMW DNA extraction for Long Read Sequencing using CTAB

DOI
dx.doi.org/10.17504/protocols.io.bp2l694yzlqe/v1
  • 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.
Benoît Vacherie
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DOI: 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 06, 2022
Protocol Integer ID: 70072
Keywords: extraction, high molecular weight, plant, leaves, DNA, Long read sequencing
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-mercaptoethanolSigma AldrichCatalog #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
Reagent15 ml conical tubes
Materials
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
Step 30
ReagentLiquid nitrogen
Materials, Step 3
ReagentIsopropanol
In Materials and 2 steps
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667
Materials, Step 7
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Materials
Reagent70% Ethanol
Step 29
ReagentGlass pasteur pipettes
Materials
ReagentEthanol 70%
Materials
ReagentTris HClP212121
Materials
ReagentChloroform
Materials, Step 9
ReagentBuffer QBTQiagenCatalog #19054
Materials, Step 19
ReagentGenomic tip 100GQiagenCatalog #10223
Materials, Step 19
Reagent1.5ml Eppendorf DNA LoBind tubes
Materials
ReagentBuffer G2QiagenCatalog #1014636
Materials, Step 17
ReagentSodium chlorideP212121
Materials
ReagentRNase AQiagenCatalog #19101
Materials, Step 6
ReagentPEG-8000PromegaCatalog #V30111
Materials
ReagentPorcelain Mortar, 145mLThermo FisherCatalog #CP1782004
Materials
ReagentVortex Mixer
Materials
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
Materials, Step 2
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
Materials
ReagentBuffer QFQiagenCatalog #19056
In Materials and 2 steps
ReagentMBP™ Wide Bore Pipette TipsThermo FisherCatalog #02707600
Materials
ReagentBuffer TE 1x
Step 33
ReagentBuffer QCQiagenCatalog #19055
Materials, Step 23
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
Materials
preparation of reagents
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
DNA Extraction
3h
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-mercaptoethanolSigma AldrichCatalog #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 ReagentChloroformContributed by users

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
ReagentIsopropanolContributed by users

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
Génomic tip purification
1h
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
DNA précipitation
3h
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 Contributed by users

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
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
Results
QC results obtained on different species.