Jun 29, 2022

Public workspaceHigh Molecular Weight Total DNA Extraction from plant tissues for Long Read Sequencing

 Forked from Total High Molecular Weight DNA Extraction from plant tissues for Long Read Sequencing
DOI
dx.doi.org/10.17504/protocols.io.14egn7yy6v5d/v1
  • 1Genome Innovation Hub, The University of Queensland
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DOI: dx.doi.org/10.17504/protocols.io.14egn7yy6v5d/v1
Protocol Citation: Subash Rai 2022. High Molecular Weight Total DNA Extraction from plant tissues for Long Read Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7yy6v5d/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: June 10, 2022
Last Modified: June 29, 2022
Protocol Integer ID: 64303
Keywords: High Molecular Weight DNA, Plant HMW DNA extraction, Neptunia HMW DNA, long read sequencing, HMW total DNA
Abstract
This protocol was developed as a research within GIH collaborative projects for a sample where a species of Neptunia leaves sample was problematic in our previously developed protocol. At step 21 of previous protocol, the solution forms either a brownish mark on the top layer of the solution or the whole solution depending on the starting sample amount which resulted brown CTAB-DNA complex. In this protocol, the problematic steps for a particular sample is improved to extract high quality High Molecular Weight (HMW) DNA >60kb. The DNA quality was assessed in Qubit, NanoDrop, TapeStation, and Oxford Nanopore Technologies. Using a LSK109 ligation chemistry and R9.4 flow cell, a total yield of 24gb with N50 29kb was generated in MinION sequencing platform.
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Guidelines
Starting materials:
  • Young and healthy tissues are ideal samples for HMW DNA extraction. The amount of sample required depends on the plant genome size. More material is required for small genome plants when compared to bigger genome plants (of equivalent sample quality).

Handling of HMW DNA:

  • Always use wide-bore pipette tips as recommended in the protocol.
  • Allow the DNA to stand in elution buffer DurationOvernight at TemperatureRoom temperature or tap the tube gently.
Note
NO vortexing at all!!

  • Avoid repeated cycle of freezing and thawing. Aliquot the required amount of DNA in multiple tubes before storing at Temperature-20 °C / Temperature-80 °C .

Citations
CITATION
Xin, Z., & Chen, J. (2012). A high throughput DNA extraction method with high yield and quality.. Plant Methods.
LINK
https://doi.org/10.1186/1746-4811-8-26

CITATION
Mayjonade, B., Gouzy, J., Donnadieu, C., Pouilly, N., Marande, W., Callot, C., Langlade, N., & Muños, S. (2018). Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules. Biotechniques.
LINK
https://doi.org/10.2144/000114460

Materials
Materials and consumables
ABC
Items descriptionCatalogue numberSuppliers/Manufacturers
Ammonium Acetate 7.5M SolutionA2706-100MLSigma Aldrich
Chloroform:Isoamyl alcohol (24:1)ACR327155000Thermo Fisher
CTAB52365-50GSigma Aldrich
Distilled water Ultra-Pure10977015Thermo Fisher Scientific
DNA LoBind tubes 1.5ml30108051Eppendorf
Dry ice--
Dynabeads M-270 Carboxylic Acid14306DThermo Fisher
EDTA (0.5M), pH-8, Nuclease-freeAM9260GLife Technologies
Ethanol (>98%)US015017Thermo Fisher Scientific
Falcon tube 15mlFAL352096In Vitro Technologies
Isoamyl alcohol (>98%)W205702-1KG-KSigma Aldrich
Liquid Nitrogen (LN2)--
P1000 wide bore pipette tips2079GPKThermo Fisher Scientific
P200 wide bore pipette tipsLC1152-965Adelab Scientific
PEG 8000V3011Promega
Proteinase K (PK) SolutionMC5005Promega
Qubit 1× dsDNA HS Assay KitQ33231Life Technologies
RNase solutionA7973Promega
Sodium Chloride71580-500GSigma Aldrich
UltraPure 1M Tris-HCI, pH-815568025Life Technologies
β-mercaptoethanolM6250-100 mLSigma Aldrich
ReagentAmmonium acetate solution for molecular biology, 7.5 MMillipore SigmaCatalog #A2706
ReagentCTAB (Hexadecyltrimethylammonium bromide)Sigma AldrichCatalog #52365-50G
ReagentUltraPure Distilled WaterThermo Fisher ScientificCatalog #10977015
ReagentDNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
ReagentDynabeads™ M-270 Carboxylic AcidThermo FisherCatalog #14306D
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentFalcon® 15 mL Polystyrene Centrifuge Tube Conical Bottom with Dome Seal Screw Cap Sterile 50/BagInvitrogenCatalog #FAL352095
ReagentART™ Barrier Specialty Pipette Tips, 1000, wide boreThermo FisherCatalog #2079GPK
ReagentPEG-8000PromegaCatalog #V30111
ReagentProteinase K (PK) Solution, 4mlPromegaCatalog #MC5005
ReagentQubit™ 1X dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q33231
ReagentRNase A Solution, 4mg/mlPromegaCatalog #A7973
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
Reagent1% β-mercaptoethanol SigmaCatalog #M6250
Reagentisoamyl alcoholSigmaCatalog #W205702
Equipment
  • Benchtop centrifuge
  • Centrifuge for 15ml falcon tube
  • Esky/container for dry ice
  • Flask Dewar or equivalent to transport LN2
  • Heat block
  • HulaMixer
  • Magnetic rack
  • Mini centrifuge
  • Mortar and pestle
  • NanoDrop
  • Qubit
  • TapeStation or equivalent
  • Thermomixer (with adapter for 15ml tubes)
Safety warnings
  • Chloroform: Isoamyl alcohol (24:1) waste should be collected in a separate waste container.
  • Experiment should be performed under fume hood after adding β-mercaptoethanol in lysis buffer during the extraction step.
  • Follow the standard Liquid Nitrogen handling procedures.
  • Consult MSDS for each required reagent and handle accordingly.
Before start
Prepare the following buffers and solutions before starting the experiment:

Lysis Buffer
AB
Tris-HCl100 mM
EDTA20 mM
CTAB (w/v)4%
NaCl1.4 M
PVP 360k (w/v)1%
β-mercaptoethanol (add just before use)]2%
Combine the reagents given in the table below.
AB
1M Tris-HCl (pH = 8)5 ml
0.5M EDTA (pH = 8)2 ml
CTAB powder2 g
PVP0.5 g
NaCl4 g
Adjust the final volume to Amount50 mL with Nuclease free water/lab grade water. Store at TemperatureRoom temperature for up to 3-4months.

High-salt TE buffer
AB
EDTA2 mM
Tris-HCl10 mM
NaCl1 M 
Combine the reagents given in the table below
AB
NaCl581 mg
0.5M EDTA (pH=8)40 μl
1M Tris-HCl (pH=8)100 μl
Complete to Amount10 mL with Distilled water Ultra-Pure. Autoclave it for long-term (1 year) storage.

Binding buffer (20% PEG8000 and 3M NaCl):
Add Amount2 g PEG 8000 and Amount1.75 g NaCl in Amount10 mL nuclease free water and mix well until it turns as a clear solution and store at cold room or 4-7°C.

Beads solution:
AB
Dynabeads™ M-270 Carboxylic Acid4%
PEG800018%
NaCl1 M
Tris-HCl pH-810 mM
EDTA pH-81 mM
  • First prepare the required volume of the solution except Dynabeads.
  • Keep the Dynabeads at RT for at least Duration00:15:00 . Mix well by vortexing, then take 4% of the beads solution (v/v) immediately.
  • Wash the beads with nuclease free water 3 times. Resuspend the beads pellet completely while washing.
  • Add the beads solution and store the beads solution at Temperature4 °C .
  • Keep the beads solution at TemperatureRoom temperature for at least Duration00:15:00 and mix well before using it.
Tissues preparation and lysis
Tissues preparation and lysis
1h 12m
1h 12m
Take Amount10 mL lysis buffer and warm it at Temperature60 °C for 15-20 min.

20m
Take ~Amount1 L of liquid nitrogen (LN2) in Dewar Flask that requires for chilling mortar and pestle and grinding the tissues.

Take dry ice in an esky/insulated container for later steps.
Grind Amount500 mg to Amount1000 mg healthy young fresh/snap frozen/frozen tissues in mortar and pestle chilled with LN2 to fine powder.
Note
It may require topping up 2-3 times LN2 while grinding the plant tissues.

15m
Keep a 15 ml falcon tube on the dry ice for Duration00:05:00 then swirl the ground powder with LN2 and pour directly into the falcon tube while keeping the falcon tube on the dry ice.

5m
Keep the lid half-opened and let LN2 to evaporate.
10m
Take out the tube and add Amount10 mL prewarmed lysis buffer (at Temperature60 °C ) with freshly added Amount200 µL β- mercaptoethanol.

1m
Pipetting
Mix well by inverting the tubes (~100 times) until the solution become more homogenous. In some sample, solution may not be homogenous but form whiteish clumps (it is normal) and incubate at Temperature60 °C in thermomixer at Centrifigation300 rpm for Duration00:30:00 .

30m
Incubation
Mix
Add Amount200 µL Proteinase K (stock conc=Amount20 mg/mL ) after 15 min of incubation.

1m
Pipetting
Mix well by inverting the tube (15-20 times) and continue the incubation.
1m
Mix
Spin the solution at Centrifigation3000 x g, Room temperature, 00:05:00 .
Note
If any clump formed during the incubation pellet would be large.

5m
Centrifigation
Take an equal volume of the supernatant in two fresh 15 ml falcon tubes using P1000 wide bore pipette tips.
3m
Pipetting
Extraction of raw HMW DNA
Extraction of raw HMW DNA
2h 37m
2h 37m
Add an equal volume of Chloroform:Isoamyl alcohol (24:1) into the solution.
30s
Pipetting
Mix the solution by inverting the tube until a milky colour appears (~100 times) and centrifuge at Centrifigation3000 x g, Room temperature, 00:10:00 .

10m
Centrifigation
Mix
Transfer the aqueous phase to a new 15ml falcon tube without disturbing interface layer.
2m
Add an equal volume of Chloroform:Isoamyl alcohol (24:1) into the solution.
30s
Pipetting
Mix the solution by inverting the tubes ~100 times and centrifuge at Centrifigation3000 x g, 00:10:00 .

10m
Centrifigation
Mix
Transfer 1 ml aqueous phase to 2 ml LoBind tube without disturbing the interface layer (much thinner than the first extraction). It requires multiple 2 ml LoBind tube.
Note
transferring aqueous phase to 2 ml tube is for making things easy for centrifugation but can be done in a single tube if the centrigugation set up is available for a bigger volume tube.

2m
Add half volume of Ammonium acetate (7.5 M). Mix well by inverting the tubes and incubate for Duration00:10:00 at TemperatureRoom temperature
12m
Pipetting
Centrifuge at Centrifigation13000 x g, Room temperature, 00:10:00 .
10m
Centrifigation
Transfer the supernatant in a fresh 2ml LoBind tube and add equal volume of Isopropanol (>98%), mix well, and incubate for Duration00:10:00 at TemperatureRoom temperature .

10m
Resuspend the pellet with Amount1 mL 70% ethanol (freshly prepared) using a wide bore P1000 pipette tip and transfer all into a 2ml LoBind DNA tube.
1m
Pipetting
Rinse the tube with additional Amount1 mL 70% ethanol to collect remaining CTAB-DNA complex. Perform the same for another 15 ml falcon tube.

1m
Wash
Incubate 2 ml tubes for Duration00:05:00 at TemperatureRoom temperature in a HulaMixer at Centrifigation9 rpm .

5m
Incubation
Spin the tube at Centrifigation13000 x g, 00:05:00 and discard the supernatant.

5m
Centrifigation
Repeat washing steps Go to & Go to once with Amount2 mL 70% ethanol.

10m
Wash
Keep the tubes under fume hood for Duration00:05:00 to remove any traces of ethanol.

5m
Resuspend the DNA pellet in Amount200 µL of prewarmed (Temperature60 °C ) High-salt TE buffer.

5m
Add Amount4 µL RNaseA and incubate at Temperature37 °C for 15-20 min.

20m
Incubation
Pipetting
Beads Purification of HMW DNA
Beads Purification of HMW DNA
1h 11m
1h 11m
Add Amount100 µL ammonium acetate (Concentration7.5 Molarity (M) ) mix well and incubate it for Duration00:10:00 at TemperatureRoom temperature . Shake it once every 5 min.
10m
Incubation
Pipetting
Mix
Spin the tube at Centrifigation13000 x g, 00:03:00 and transfer the supernatant using wide-bore pipette tips into a fresh tube.

3m
Centrifigation
Add equal volume of binding buffer and mix well by inverting the tube.
1m
Pipetting
Mix
Add Amount150 µL beads solution (8-9 million beads) and incubate it for Duration00:30:00 at TemperatureRoom temperature in a HulaMixture.

30m
Incubation
Pipetting
Place the tube in magnetic rack for 2-3 min and remove the supernatant and wash the beads with Amount500 µL freshly prepared 70% ethanol (clumping of beads may appear but try to dislodge by inverting the tube several times).

3m
Wash
Wash the pellet once again with Amount500 µL freshly prepared 70% ethanol.

Wash
Take out the tubes from magnetic rack and add Amount500 µL freshly prepared 70% ethanol.
30s
Dislodge the beads by inverting the tubes.
30s
Place the tubes back to the magnetic rack for 2 min and remove the supernatant.
2m 30s
Add Amount75 µL prewarmed (at Temperature50 °C ) Concentration10 millimolar (mM) Tris-HCl (elution buffer) Ph8 and incubate at TemperatureRoom temperature for Duration00:15:00 .

15m
Incubation
Pipetting
Place the tube back in the magnetic rack and leave it for Duration00:05:00 .

5m
Remove the supernatant in the fresh 1.5ml LoBind DNA tube.
Note
If the eluate is very viscus and beads could not pellet either add more elution buffer or centrifuge Centrifigation13000 x g, 00:05:00 .


30s
Assess DNA quality in NanoDrop, Qubit, and TapeStation/PFGE
Worked Results
Worked Results




General Quality check:

QC check in Oxford Nanopore Sequencing:

Read length distribution ( estimated N50 = 29.5kb) in MinION Nanopore sequencing. DNA was extracted from frozen leaves sample. The size selection was performed with standard SRE kit (circulomics) prior to the library preparation using ONT LSK109 kit.




Citations
Mayjonade, B., Gouzy, J., Donnadieu, C., Pouilly, N., Marande, W., Callot, C., Langlade, N., & Muños, S.. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules
https://doi.org/10.2144/000114460
Xin, Z., & Chen, J.. A high throughput DNA extraction method with high yield and quality.
https://doi.org/10.1186/1746-4811-8-26