Jul 03, 2023
Version 2
High-Molecular-Weight SPRI-aided DNA extraction from Mimulus (Phrymaceae) leaf tissue V.2
- 1Tulane University
Protocol Citation: Bolívar Aponte Rolón 2023. High-Molecular-Weight SPRI-aided DNA extraction from Mimulus (Phrymaceae) leaf tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xn8rlqe/v2
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 30, 2023
Last Modified: July 03, 2023
Protocol Integer ID: 84324
Keywords: fungi, mimulus, high-molecular-weight, dna, endophytes, magnetic, spri, miseq, illumina
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Abstract
This protocol is adapted from Russo et al. 2023 methods for high-molecular weight DNA extraction. I modified the protocol to use reagents and incubation conditions used by the Ferris Lab at Tulane University to extract DNA from Mimulus spp. (monkeyflower) tissue (leaf and buds), see protocol. The original CTAB:Chloroform protocol used by the Ferris lab traces back to the Willis Lab at Duke University. The objective of these DNA extractions are to collect fungal DNA present in leaf tissue. I have incorporated sample preparation procedures used by the Arnold Lab at The University of Arizona to eliminate contamination of samples and optimize DNA extractions from preserved leaf tissue for future fungal ITS Illumina MiSeq sequencing. Solid phase reversible immobilization beads (SPRI) utilized are adapted from Rohland and Reich 2012 and Liu et al 2023. All extractions were performed un a sterile biosafety cabinet to prevent sample contamination.
Prior to being placed in CTAB, photosynthetic tissues from plants were surface sterilized with sequential washes in 95% EtOH (10 s) , 0.5% sodium hypochlorite (1 min), and 70% EtOH (1min) and air dried under sterile conditions. Due to the small size of monkeyflower plants, the maximum amount of leaf tissue produced per host were placed in 750- 1000 µL of CTAB buffer and kept at RT until DNA extraction process.
*Several aspects of this protocol, mainly those with reagent amounts and molarities, have changed compared to the Russo et al 2023 protocol and the Willis Lab standard CTAB extraction protocol from ca. 2010.
Guidelines
General Notes
DNA Extractions for Illumina Seq. are the most sensitive. If you need high quality low contamination DNA that is high molecular weight, make sure to use the gentler methods of mixing. Use wide bore pipet tips to help reduce shearing of DNA. Do not vortex any samples to mix, just invert or gently tap/flick tubes. To reduce contamination, be sure to check that all samples come out of the first chloroform step with a clear aqueous layer, not cloudy. Sodium chloride (5M NaCl) helps reduce polysaccharide contamination (compared to the Isopropanol precipitation without the added NaCl). High salt concentrations also aid in DNA precipitation, so you should get more DNA precipited and cleaned with chloroform wash.
Sample Handling
- Gloves must be worn at all times. Change gloves frequently and decontaminate often with DNA Away.
- Pipetting must be done extremely carefully to minimize the risk of aerosols that can easily cause contamination between samples.
- Use only unopened, sterile, aerosol-resistant pipette tips (filter tips) to minimize contamination of the pipette shaft and your samples. Clean the body of the pipettes regularly with DNA Away.
- Discard pipette tips after each use to avoid cross-contamination. Eject pipette tips carefully to prevent aerosol formation or other splashing.
- Remember to always clean the hood and any equipment with DNA Away and decontaminate where possible with UV for 30 minutes prior to use to avoid cross-contamination.
- The pipettes used need to be calibrated annually.
- Be careful not to touch the inside lid of tubes as it will cross-contaminate samples.
Materials
Equipment
- Centrifuge capable of 24 tubes, 10,000 rpm or 96 well-plate at 4,000 rpm
- Class II Biosafety Cabinet
- Stainless stee; beads 3.2mm Next AdvanceCatalog #SSB32
- Stainless stell bead blend 0.9-2mmNext AdvanceCatalog #SSB14B
- Ziconium oxide beadsNext AdvanceCatalog #ZROB20-RNA
- Liquid Nitrogen
- Water Bath (that can be set to 60°C)
- Benchtop shaking incubator (Labnet 222DS)
- Heating block (able to set at 50°C)
- Eppendorf or Rainin Pipettes: 1000 µL, 200 µL, and 20 µL
- 1.5/2ml tube magnetic rack.
Reagents and consumables
- 2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
- Polyvinylpyrrolidone (PVP-40)VWR InternationalCatalog #0507
- 5M NaCl solution (Make ahead and autoclave if there isn’t any in the cabinet)
- 10 mM Tris-HCL pH 8.0 or
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)
- 95% Ethanol
- 80% Ethanol (frozen at -20°C)
- DNase Away
- 10 % sodium hypochlorite solution (Bleach)
- CTAB bufferOPS DiagnosticsCatalog #CEB 500-02
- Pipette tips: 1000 µL (×4), 200 µL (×2), and 20 µL (×1)
- Molecular grade water (100 mL)
- Screw cap micro tubes 2 mLThermo ScientificCatalog #3469NK
- Eppendorf DNA low-bind microtube 2 mL Fisher ScientificCatalog #022431048
Protocol materials
Stainless stell bead blend 0.9-2mmNext AdvanceCatalog #SSB14B
Materials, Step 9.2
Ziconium oxide beadsNext AdvanceCatalog #ZROB20-RNA
Materials, Step 9.3
Polysorbate 20 (Tween-20)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416
Step 29
Fresh 80% Ethanol
Step 34
Polyvinylpyrrolidone (PVP-40)VWR InternationalCatalog #0507
In Before starting, Materials, Step 16
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)
In Materials and 2 steps
CTAB bufferOPS DiagnosticsCatalog #CEB 500-02
Materials, Step 16
Eppendorf DNA low-bind microtube 2 mL Fisher ScientificCatalog #022431048
In Materials and 2 steps
Stainless stee; beads 3.2mm Next AdvanceCatalog #SSB32
Materials, Step 9.1
SeraMag SpeedBeads Carboxyl-Magnet-Beads hydrophobFisher ScientificCatalog #GE Healthcare 65152105050250
Step 29
Screw cap micro tubes 2 mLThermo ScientificCatalog #3469NK
In Materials and 2 steps
10 mM Tris-HCL pH 8.0
In Materials and 2 steps
2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
Materials, Step 16
RNase A 10mg/ml, DNase and Protease-free Thermo ScientificCatalog #EN0531
Step 19
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884
Step 29
Sodium ChlorideFisher ScientificCatalog #S271
Step 21
Polyethylene glycol (PEG-8000)Fisher ScientificCatalog #FLBP2331
Step 29
Safety warnings
Careful handling all reagents, specially CTAB and Chlorofom:IAA
Before start
Make sure to…
- Turn on the water bath to 60°C and heating block to 50°C.
- Get liquid nitrogen.
- Get sample tubes/plates from the -80°C or 20°C freezer.
- Keep samples frozen! (On dry ice).
If and only if…
You have a fresh homemade CTAB buffer that does not have polyvinylpyrrolidoneand β-mercaptoethanol will you add
these reagents to stock buffer.
Instructions
- Right before use of CTAB buffer; prior to starting extraction, add polyvinylpyrrolidone and β-mercaptoethanol.
- Dissolve 1.1 – 2.2 grams of Polyvinyl-Pyrrolidone (PVP) per each 50-100ml CTAB (1.1 – 2.2% of the total buffer vol.).
Once these have been added the shelf life of the buffer is only 2-3 days.
- If tissue is in homemade CTAB buffer and no freeze drying/lyophilization was performed (jumped directly to DNA extraction) you can add 2.25 µL of β-mercaptoethanol per 700 µL L CTAB, as well as 8 -16 µL of Polyvinylpyrrolidone (PVP-40)VWR InternationalCatalog #0507 (1.1-2.2 % solution) per 700 µL CTAB.
DO EXTRACTIONS IN THE BIOSAFETY CABINET (except for grinding, and spinning steps).
Sample preparation
Sample preparation
Clean the biosafety cabinet, pipettes, tip boxes and any other instrument to be used with 70% EtOH, 10 % Bleach and 95 % EtOH. Surface sterilize all instruments in biosafety cabinet with UV light for 00:30:00 .
30m
Transfer 0.15-0.3 grams of leaf tissue to new 2 mL tube. Leaving remaining leaf tissue in original 15 mL vial tube.
Note
This step depends on how easy to handle your plant tissue is. Fresh tissue is easier to handle. Mimulus spp. tissue preserved in CTAB turns "mushy" and it is difficult to handle and transfer.
Screw cap micro tubes 2 mLThermo ScientificCatalog #3469NK
Weigh empty 2 mL tube to determine how much tissue you need to place in tube.Screw cap micro tubes 2 mLThermo ScientificCatalog #3469NK
Weigh 2 mL tube with leaf tissue to determine if it has the correct amount.
Note
Samples with more tissue (> 0.3 <0.6) can be split into two tubes.
Quickly after placing tissue in tubes, place tube cap and close tightly.
Note
Don’t let samples sit at room temperature for very long because the DNA will degrade.
Place tubes in the -20 °C or -80 °C freezer. Until ready for freeze drying/lyophilization step.
Freeze drying/Lyophilization
Freeze drying/Lyophilization
2d 1h
2d 1h
Once all your samples are cold, turn on the refrigerator for the lyophilizer and place the glass plate on the front.
Note
Make sure there is no water in the chamber or drain tube.
Once the lyophilizer temperature is at -40 °C , turn on the vacuum pump.
Note
Vacuum pressure must be below 150 milliTorr . If pressure doesn't drop within the hour do not use machine.
1h
When the pressure is at the appropriate level, release the pressure by turning the white port 180 degrees.
Note
Make sure there is a vacuum seal, and the pressure returns to <150 milliTorr .
Quickly take frozen tubes out of the freezer and place inside the lyophilizer chamber. Close the lid and close the port.
Lyophilize samples for a minimum of 48:00:00 .
Note
To remove the samples, first turn the white port on the lyophilizer 180 degrees to release the pressure in the chamber. Once the pressure is released and the gauge reads 0, turn off the vacuum pump and then the refrigerator. Remove the glass plate from the front and wipe out the water after it has melted (also shake out the drain tube).
2d
Pulverization
Pulverization
In a sterile laminar flow hood, remove the parafilm or lid from the tubes (being careful not to touch the inside lip of the cap) and add beads for homogenization. For all plant samples except mosses, add the following:
Note
The pulverization/homogenization of tissue can be performed beforehand and samples stored away until DNA extraction. I prefer to pulverize all my samples, store them and work my way through the DNA extraction.
Add 100 μL (2 beads) of the autoclaved 3.2 mm stainless steel beads.Stainless stee; beads 3.2mm Next AdvanceCatalog #SSB32
Note
Beads can also be added at the same time sample tissue is weighed and transffered to 2 mL tubes. Organize your workflow as you see fit.
Add 2-3 of the autoclaved 2 mm zirconium oxide beads to each tube with a sterile scoop.Stainless stell bead blend 0.9-2mmNext AdvanceCatalog #SSB14B
Add 100 μL of the autoclaved stainless steel bead blend, (0.9-2.0mm) using a sterile scoop. Close the lids securely.
Ziconium oxide beadsNext AdvanceCatalog #ZROB20-RNA
Tubes with beads can be placed in a -20 °C freezer until ready to extract DNA.
11. Dip tubes/96-well plates in liquid nitrogen.
Note
a) Make sure there are no cracks in the plastic and that the plate has its bottom panel to keep the tubes and racks from shattering.
b) Clamp plates into shaker rack in Geno Grinder 2010 or TissueLyser LT. Always clamp plates into both shaker racks and make sure tubes are evenly distributed across plates.
12. Shake at 1500 strokes/minute for 3 minutes in GenoGrinder 2010 or 25-30 Hz for 3 minutes if using TissueLyzer LT.
Equipment
GenoGrinder 2010
NAME
SPEX-SamplePrep
BRAND
SP 2010-115
SKU
LINK
High-throughput plant & animal tissue homogenizer and cell lyser.
SPECIFICATIONS
Equipment
Tissuelyser LT
NAME
Qiagen
BRAND
85600
SKU
LINK
Tissue and cell lyser
SPECIFICATIONS
Note
a) Decrease or increase time as necessary.
b) Check periodically and do not grind for longer than necessary. Do not let tissue thaw, if you notice melting, re-dip in liquid N2.
c) When tissue is ground to a fine powder, dip briefly into liquid nitrogen again to keep frozen.
d) Examine tubes carefully to ensure proper pulverization. Repeat if necessary.
Centrifuge plate fast and briefly at 4000 rpm when using 96-well plate centrifuge or 10.000 rpm when using Corning LSE centrifuge to get powder off lids.
Note
Anywhere from 1-3 minutes depending on how much the sample adheres to the lid of the tube.
Store in -20 °C or -80 °C until DNA extraction steps.
DNA Extraction
DNA Extraction
30m
30m
Surface sterilize all workbench surfaces and instruments with a 10% solution of 0.5% sodium hypochlorite, 70% EtOH and 95% EtOH prior to placement in biosafety cabinet. Surface sterilize all
instruments in biosafety cabinet with UV light for 00:30:00 .
30m
Make Extraction Master Mix (Pre-warm to 60°C):
Measure out700 µL of CTAB extraction buffer per sample into 15-50 mL conical centrifuge tube. Add2.25 µL β-mercaptoethanol per 700 µL of CTAB extraction buffer to the same conical tube (0.3% of the total extraction buffer volume).
Basic recipe:
[(# of samples x 1.1) x 700 µL of CTAB] + [(# of samples + 1) x 2.25 µL β-mercaptoethanol]
Note
See Guidelines and Warning for more information.
2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
CTAB bufferOPS DiagnosticsCatalog #CEB 500-02
Polyvinylpyrrolidone (PVP-40)VWR InternationalCatalog #0507
Add 700 µL of preheated Extraction Master Mix to each tube, replace the lids (if using strip tubes).
Note
a) Shake tubes by hand vigorously until tissue is thawed and fully suspended in the buffer. Dip plate or tubes in hot water bath to assist the thaw if needed.
b) If fine tissue powder is collected at the bottom and it does not mix well with CTAB buffer, then place in Vortex 5-30 seconds (depending on tissue) or TissueLyser LT for 1 minute at 15 Hz.
Beware of DNA shearing.
Note
Always make sure to include an extraction blank (negative control).
Incubate tubes for 00:20:00 in a 60 °C water bath.
Note
Important: Invert tubes/plates gently about 10 times every ~10 minutes during incubation.
If using strip tubes: Keep costar plate lid off during water bath and grinding step (prior) and refasten strip tube caps before inverting – they will pop off! Keep fingers firmly on strip tube lids while inverting.
20m
After 20 minutes incubation in step 18, add 4 µL of RNase A.
RNase A 10mg/ml, DNase and Protease-free Thermo ScientificCatalog #EN0531
Incubate at 60 °C for 00:10:00 .
Note
Invert 10 times once during incubation.
10m
All 200 µL of 5M NaCl (or 5M Sodium/Potassium acetate). To fully precipitate proteins and polysaccharides in extraction buffer.
Note
Slowly pipette up and down or invert 25 times.
Sodium ChlorideFisher ScientificCatalog #S271
Add 900 µL of chloroform/isoamyl (24:1) (1 volume) to each tube.
Place tubes in a nutating mixer for 00:10:00 at 24 rpm.
Note
a) If possible, use instruments that can produce 10-20 rpms. Less shaking = less shearing.
b) Can invert by hand 20 - 40 times, instead of shaking, to further
decrease shear. Wear extra thick nitrile gloves to prevent chloroform
exposure to skin. If you get chloroform on your gloves, remove them as soon as
possible and discard them in the solid hazardous waste bag in the hood!
Equipment
S0500 Mini Nutating Mixer
NAME
VWR
BRAND
82007-202
SKU
LINK
Mixer
SPECIFICATIONS
10m
Centrifuge for 00:10:00 at 4000rpm when 96-well plate centrifuge or 00:10:00 at10.000 rpm when using Corning LSE centrifuge.
Note
A band of tissue debris will separate the aqueous (upper) and chloroform/organic (lower) layers. The top aqueous layer should be clear, not cloudy. If cloudy, go back and re-do steps 23 and 24.
20m
Carefully pipette off ~800 µL -900 µL of the top aqueous layer using filter pipet tips and
transfer to sterile new tubes or 96-well plate.
Note
Avoid drawing up debris or chloroform from the middle and lower layers (if so, re-centrifuge briefly and transfer again).
Eppendorf DNA low-bind microtube 2 mL Fisher ScientificCatalog #022431048
Repeat steps 22-24 go to step #22 .
Note
This will remove any leftover RNase and contaminants.
After repeating steps 22-24. Carefully pipette ~700 µL -800 µL of the top aqueous layer using wide-bore filter pipet tips and transfer to sterile new tubes or 96-well plate.
Note
a) Avoid drawing up debris or chloroform from the middle and lower layers (if so, re-centrifuge briefly and transfer again).
b) You can cut 3-4 mm of P1000 tips to make yourself "wide-bore" tips. Do this beforehand in a sterile environment or autoclave tips afterwards.
Eppendorf DNA low-bind microtube 2 mL Fisher ScientificCatalog #022431048
Note
Feel free to pause and get yourself ready for DNA purification. DNA is stable at this stage and can be left at room temperature while you prepare.
SPRI aided isolation and purification of DNA
SPRI aided isolation and purification of DNA
45m
45m
Turn ON heating block to 50 °C and place 10 mM Tris-HCl or 1X TE buffer to pre-heat.
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA) 10 mM Tris-HCL pH 8.0
Add SPRI bead mixture according to the following:
Volume of SPRI beads to add = 1.2 × DNA volume (from step 27)
Note
Adjust the ratio of SPRI beads as you see fit. Russo et al 2023 use 0.7x. See Rohland and Reich 2013 and Liu et a 2023 for SPRI protocol and
| A | B | C | D | |
| Final concentration for a 1.25% SPRI bead solution for use in DNA extraction process. | ||||
| Reagent | 10 mL | 50 mL | Final Concentration | |
| Sera-Mag Speed Beads | 125 µL | 625 µL | 1.25% | |
| PEG-8000 | 2.5 g | 12.5 g | 25% | |
| 5 M NaCl | 1000 µL | 25 mL | 2.5 M | |
| 1 M Tris-HCl (pH 8.00 | 100 µL | 500 µL | 10 mM | |
| 0.5 M EDTA (pH 8.00 | 20 µL | 100 µL | 1 mM | |
| 10% Polysorbate 20 (Tween 20) | 5.5 mL | 275 mL | 10 % | |
| Nuclease free H2O | fill to 10 mL | fill to 50 mL | n/a | |
SeraMag SpeedBeads Carboxyl-Magnet-Beads hydrophobFisher ScientificCatalog #GE Healthcare 65152105050250
Polyethylene glycol (PEG-8000)Fisher ScientificCatalog #FLBP2331
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884 Polysorbate 20 (Tween-20)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416
Incubate for 00:30:00 in a nutating mixer at 24 rpms (default).
30m
Spin down for 00:00:05 on centrifuge.
5s
Place the tubes on a magnetic rack for 00:05:00 or wait until the
solution is clear.
Note
This can take several minutes depending on the viscosity and type of sample. Wait until the solution is clear before proceeding to the next step.
5m
Aspirate the cleared solution from the tubes, inspect pipette tips for bead residue, and discard.
Note
This step must me performed while the tubes are placed on the magnetic rack. Avoid disturbing the settle magnetic beads. If beads are drawn into tips, leave behind a few microliters of solution..
Dispense 1 mL of fresh 80% ethanol to each tube and invert 25 times to resuspend bead pellet.
Note
Flick tubes gently to help separate the beads from the tube wall. Avoid prolonged incubation in EtOH.
Fresh 80% Ethanol
Spin down for 1-2 s on centrifuge.
2s
Aspirate out the ethanol, inspect pipette tips for bead residue, and discard.
Repeat for a total of three washes. go to step #33
Let beads air-dry for 00:01:00 after final wash.
1m
Take tubes out of the magnetic rack, add 50 μL of elution buffer (10 mM TRIS-HCl pH 8.0,
or 1X TE buffer) pre-heated to50 °C to each tube and pipette mix 5 times.
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)Contributed by users 10 mM Tris-HCL pH 8.0Contributed by users
Spin down for 1-2 s on centrifuge.
2s
Incubate tubes at 37 °C for 00:15:00 in benchtop shaking incubator at 20 rpm.
Note
If possible, use instrument that can produce 10-20 rpms.
15m
Place tubes in magnetic rack and let sit for 00:15:00 -00:30:00 .
Note
I've had best results with 30 minutes.
45m
Collect ~50 µL of supernatant and transfer to a new tube.
Place in tube stand and let sit at RT overnight.
Note
This will allow the DNA to homogenize and relax. You can relax too!
12h
Store DNA at -20 °C or -80 °C until ready for quantification with Qubit or PicoGreen assay and downdtreams analyses.
Protocol references
Russo A, Mayjonade B, Frei D, Potente G, Kellenberger RT, Frachon L, Copetti D, Studer B, Frey JE, Grossniklaus U and Schlüter PM (2022) Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families. Front. Plant Sci. 13:883897. doi: 10.3389/fpls.2022.883897
Liu, D., Li, Q., Luo, J. et al. An SPRI beads-based DNA purification strategy for flexibility and cost-effectiveness. BMC Genomics 24, 125 (2023). https://doi.org/10.1186/s12864-023-09211-w
Rohland N, Reich D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 2012 May;22(5):939-46. doi: 10.1101/gr.128124.111. Epub 2012 Jan 20. PMID: 22267522; PMCID: PMC3337438.
Jana M. U'Ren, A. Elizabeth Arnold 2017. 96 well DNA Extraction Protocol for Plant and Lichen Tissue Stored in CTAB. protocols.io https://dx.doi.org/10.17504/protocols.io.fscbnaw