Aug 28, 2024
Wheat leaf high-molecular weight (HMW) DNA extraction
- Maximillian RW Jones1,
- Nikolai Adamski1,
- Lorelei Bilham2,
- Cristobal Uauy1
- 1John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom;
- 2School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
- Uauy Lab - JIC
Protocol Citation: Maximillian RW Jones, Nikolai Adamski, Lorelei Bilham, Cristobal Uauy 2024. Wheat leaf high-molecular weight (HMW) DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xpy1lqe/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
Created: November 23, 2023
Last Modified: August 28, 2024
Protocol Integer ID: 91372
Keywords: HMW, high-molecular weight, DNA extraction, wheat, plant, nuclei extraction, cereal, nuclei, pacbio
Funders Acknowledgement:
UK Biotechnology and Biological Sciences Research Council (BBSRC)
Grant ID: BB/X011003/1
UK Biotechnology and Biological Sciences Research Council (BBSRC)
Grant ID: BB/X01102X/1
European Research Council
Grant ID: 866328
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Abstract
This protocol is for the extraction of high-molecular weight DNA from wheat leaf tissue. The product is suitable for long-read sequencing such as PacBio. The protocol may also be effective for leaves of other cereal species. For example, we have had success using it with tef (Eragrostis tef).
The protocol consists of four phases; 1) buffer preparation, 2) harvesting of leaf tissue, 3) crude nuclei extraction, and 4) HMW extraction. The last of these uses the PacBio Nanobind® plant nuclei kit.
Using this protocol we have produced good quality DNA extractions with the following features:
- Fragment size distribution peak >50 kb (Agilent Tapestation, Genomic DNA ScreenTape)
- Concentration between 350 and 600 ng / µl (Invitrogen Qubit 3 fluorometer)
- 260/280 ratio 1.75-1.90 and 260/230 ratio 1.75-1.90 (Thermo Scientific Nanodrop)
This protocol was originally adapted from Dvorak et al., 1988, but has been modified over many years.
Materials
Buffer and filter preparation
Equipment
- Precision balance
- Magnetic stirrer and flea(s)
- pH meter
- Scissors
- Permanent marker pen
Consumables / reagents
- Spermine (powder)
- Spermidine (neat liquid)
- EDTA
- Tris-HCl pH 8.0 solution
- KCl
- 10 M NaOH
- Sucrose crystals/powder
- Triton X-100
- Miracloth
- Cheesecloth
Tissue collection
Equipment
- Scissors
- Forceps
- Dewar containing liquid nitrogen (if freezing tissue)
- Permanent marker pen
- Balance (0.1 g precision)
Consumables/reagents
- 50 mL Falcon tubes (or equivalent)
- 70% ethanol in spray bottle
- Paper towels
Nuclei extraction
Equipment
- Fume hood
- Small food processor or jug blender (not a hand/stick blender)
- Three or four 2 L buckets of ice
- P1000 pipette
- P5000 pipette
- 250 mL centrifuge bottles
- Large plastic funnel
- 100 mL measuring cylinder
- Glass rod or blunt forceps
- Scissors
- Small paint brushes (<1/4 width of a microfuge tube)
- Cup of soapy water for used paint brushes
- Timer
- Two 1 L waste beakers
- Chemical sharps bucket
- Solid chemical waste bin
- Permanent marker pen
Consumables / reagents
- β-mercaptoethanol
- 70% ethanol in spray bottle
- 2 mL Eppendorf Protein LoBind tubes (catalog no. 0030108132 or 0030108450)
- 1000 µL tips
- 5000 µL tips
- Paper towels
HMW DNA extraction
Equipment
- Magnetic tube rack
- Vortexer
- Tabletop/mini centrifuge
- Micro centrifuge
- Heat block or water bath
- Microvolume spectrophotometer (e.g. Thermo Fisher Nanodrop)
- P200 pipette
- P1000 pipette
- Chemical sharps bucket
- Permanent marker pen
Consumables / reagents
- PacBio Nanobind® plant nuclei kit (SKU 102-302-000, previously NB-900-801-01)
- 2 mL Eppendorf Protein LoBind tubes (catalog no. 0030108132 or 0030108450)
- 100% isopropanol (2-propanol)
- 96-100% ethanol
- 200 µL nuclease-free tips
- 1000 µL nuclease-free tips
Prepare 10x HB
| Reagent | Final conc. | e.g. for 1 L | |
| spermine crystals | 10 mM | 3.425 g | |
| spermidine liquid | 10 mM | 2.545 g | |
| EDTA | 100 mM | 200 mL of 0.5 M solution | |
| Tris-HCl pH 8.0 solution | 100 mM | 100 mL of 1.0 M solution | |
| KCl | 800 mM | 267 mL of 3.0 M solution |
- Dissolve / mix above components into 75% desired final volume of deionised H2O
- Adjust pH to 9.4-9.5 with 10 M NaOH
- Fill to final volume with deionised H2O
- Store at 4 °C for up to 6 months
Prepare 1x HB
| Reagent | Final conc. | e.g. for 2 L (~12 samples) | |
| 10x HB | 1x | 200 mL | |
| sucrose | 500 mM | 342.3 g |
- Dilute 10x HB 10-fold with deionised water
- Dissolve sucrose via magnetic stirrer
- Store at 4 °C for up to 3 months
Prepare H+20 buffer
| Reagent | Final conc. | e.g. for 500 mL (~100 samples) | |
| 10x HB | 1x | 50 mL | |
| sucrose | 500 mM | 85.6 g | |
| 1x Triton X-100 | ~0.2x | 100 mL |
- Dilute 10x HB to 3/5 final volume
- Dissolve sucrose via magnetic stirrer
- Top up to 4/5 final volume with de-ionised water
- Reduce stirring speed and add Triton X-100 slowly until final volume is reached. Slow addition and slow stirring is necessary to prevent excessive foaming.
- Store at 4 °C for up to 6 months
Prepare filtration sandwiches
Prepare squares of Miracloth sandwiched between two sheets of cheesecloth. These should be large enough to line your plastic funnel.
Safety warnings
β-mercaptoethanol: can be toxic if ingested, and fatal if inhaled or absorbed through the skin. Vapours can irritate the eyes, mucous membranes, and respiratory tract. Symptoms of inhalation exposure may include coughing, sore throat, and/or shortness of breath. Work under a fume hood where indicated in the protocol.
Before start
Please see the Materials section for instructions on buffer preparation, storage conditions, and longevity.
We highly recommend you read through the whole protocol before attempting it as certain steps are complex and time-critical.
Tissue collection
Tissue collection
Sanitise scissors and/or other tools, plus gloved hands, with 70% ethanol. Collect 2-5 g leaf blade tissue into a 50 mL Falcon tube. Do not use leaf sheath tissue. Avoid using diseased, bleached, or senescing sections. According to PacBio, younger leaves generally produces higher DNA quality and quantity. Avoid bruising tissue as much as possible. Using forceps may help with this. Re-sanitise tools and hands between samples.
If conducting nuclei extraction immediately, bury Falcon tube up to cap in ice.
If conducting nuclei extraction at a later date, snap freeze the Falcon tube in liquid nitrogen then store at -70 to -80 °C.
Crude nuclei extraction
Crude nuclei extraction
Set up fume hood with the following:
- Blender (~1 L volume, preferably glass-walled as some plastics may react with β-mercaptoethanol)
- Tissue samples on ice
- 1xHB on ice (100 mL per sample)
- H+20 buffer on ice (5 mL per sample)
- β-mercaptoethanol (400 µL per sample)
- 250 mL conical centrifuge bombs on ice (1 per sample) (e.g. Corning CLS430776) (do not use flat-bottomed centrifuge bottles)
- Mira/cheese-cloth filtration sandwiches (1 per sample)
- Large plastic funnel
- 100 mL graduated cylinder
- Glass rod
- Scissors
- Timer
- 70% ethanol for cleaning
- Blue roll
- 1 L waste beaker
If using frozen tissue, finish preparing the fume hood before removing samples from freezer.
Set up centrifuge with correct rotor and swing bucket attachments and begin cooling
to 4 °C. Place 5 mL of H+20 buffer into a 250 mL centrifuge bottle per sample. Label accordingly. Place
on ice in an ordered manner.
Working in the fume hood, add 640 µL β-mercaptoethanol to 160 mL of 1xHBS for each sample and mix well. This mixture is referred to as 1xHB/β-ME for the remainder of this protocol.
Place funnel into one of the 250 mL centrifuge bombs, still on ice. Insert the Mira/cheese-cloth sandwich and push down with the glass rod or gloved finger.
Measure out 100 mL 1xHB/β-ME into the blender (for 2-5 g tissue - if using more tissue increase to 150 mL)
Cut plant tissue into small pieces directly into the blender. Start blending on a low setting until the plant tissue is incorporated into suspension. Then blend on high for one minute.
Filter the ground sample through the filtration sandwich into the centrifuge bomb. While this is dripping through, place blender under a fast-flowing tap, being sure to also rinse the lid. After filtration is complete, squeeze out remaining sample liquid from cloth into the waste beaker and discard cloth into a chemical waste bin. Thoroughly rinse the funnel and your gloved hands. Wipe scissor blades with a paper towel soaked in 70% ethanol. Cap the centrifuge bomb, gently shake, and place back on ice.
Repeat until all samples done. Samples should be incubated on ice for at least 10 minutes before moving onto Step 14.
Spin samples for 20 minutes at 1500 x g at 4 °C. If necessary for balance, adjust volumes with iced 1xHB/β-ME and/or use H2O-filled balance bombs.
While samples are spinning, tidy the fume hood and re-prepare it with the following:
- 1xHB/β-ME on ice
- Additional ice buckets with enough space for all samples
- Five-layer stacks of paper towels for blotting sample bottles
- Small paint brushes soaking in 1xHB
- P1000 pipette
- 1000 µL tips with cut ends
- 1 L waste beaker
For each sample bottle, carefully pour off the supernatant into the waste
bucket, then invert onto the stacked paper towels to remove excess liquid.
Add 1 mL of ice-cold 1xHB/β-ME to each centrifuge bottle. Use a clean paint brush (soaked in 1xHB) to resuspend the pellet. Next, mix using a P1000 pipette using a cut-tip until solution is homogeneous. Transfer sample to a 50 mL pre-labelled Oakridge tube.
Bring the volume of each sample to approximately 15 mL using ice-cold 1xHB/β-ME. Spin for 10 min at 1100 x g at 4°C.
Repeat Steps 15 to 17 twice more. The supernatant should be transparent after the
last centrifugation. The same paint brush can be re-used for the same sample.
Decant the supernatant into the waste beaker and add 1 mL of ice-cold 1XHB to the pellet per gram of tissue being processed (e.g. 3 mL for 3 g tissue).
Resuspend the pellet using a fresh paint brush pre-soaked in ice-cold 1XHB. Mix with a P1000 pipette using a cut-tip until the solution is homogeneous.
Transfer each mL of the nuclei suspension into a fresh 2 mL Protein LoBind microcentrifuge tube (equivalent to 1 g tissue/tube).
Spin the 2 mL tubes for 5 min at 7,000 x g at room temperature.
Either proceed directly to Step 24, or snap freeze the nuclei pellets in liquid nitrogen and store at -70 to -80 °C to continue the protocol at a later date. We have only tested using frozen nuclei pellets up to 1 week old, but they may be viable for longer.
HMW DNA extraction
HMW DNA extraction
For the extraction of HMW DNA from the nuclei prep, we utilised PacBio's Nanobind® plant nuclei kit and protocol with one minor modification.
Overview:
Protocol:
Modifications:
- Use 2 mL rather than 1.5 mL Protein LoBind tubes. The more rounded bottom prevents the Nanobind disks becoming lodged.
Protocol references
Jan Dvorak, Patrick E. McGuire, and Brandt Cassidy. 1988. Apparent sources of the A genomes of wheats inferred from polymorphism in abundance and restriction fragment length of repeated nucleotide sequences. Genome. 30(5): 680-689. https://doi.org/10.1139/g88-115