Jul 23, 2018
Plant nuclear genomic DNA preps
- 1Plant and Food Research
- High molecular weight DNA extraction from all kingdomsTech. support email: See@each.protocol
Protocol Citation: Elena Hilario 2018. Plant nuclear genomic DNA preps. protocols.io https://dx.doi.org/10.17504/protocols.io.rncd5aw
Manuscript citation:
Naim F, Nakasugi K, Crowhurst RN, Hilario E, Zwart AB, Hellens RP, et al. (2012) Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein. PloS one 7: e52717
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: July 13, 2018
Last Modified: July 23, 2018
Protocol Integer ID: 13732
Keywords: nuclei, plant, high molecular weight DNA, PFGE
Abstract
The isolation of nuclear genomic DNA of high molecular weight is becoming a crucial step for obtaining long read sequencing data produced by the PacBio and Oxford Nanopore platforms. Although it involves a few more steps than a standard total genomic DNA preparations, it is worth optimizing the protocol to avoid wasting valuable sequence data on organellar DNA. The current protocol has been successfully applied to kiwifruit, apple, mānuka, Nicotiana benthamiana, some solanaceas, and two insects (pupae of the light brown apple moth (Epiphyas postvittana) and hind leg of a weta (Deinacrida spp)). Three alternative methods to extract DNA are also presented.
Guidelines
Each plant poses a different challenge regarding the best approach to extract good quality DNA. The following review gives a very thorough introduction on this topic: Kasem S, Rice N, Henry R (2008) DNA extraction from plant tissue. Plant Genotyping II: SNP Technology. pp. 219-271.
The method presented here has been applied in a mid-throughput manner using a blender to homogenize the plant tissue instead of the traditional freeze/grind method with liquid nitrogen. However if the leaf tissue is tough (for example mānuka or rosemary) it would be best to use liquid nitrogen and a precooled mortar and pestle. The same would apply to very precious samples where nothing should be wasted.
Safety warnings
The protocol requires β-mercaptoethanol, chloroform and guanidine hydrochloride (Qiagen DNeasy Plant Maxi kit only). Dispose these chemicals according to your safe method of use and local regulations.
Before start
Solutions and Reagents
- NEB (Nuclei Extraction Buffer) 0.5 M Mannitol, 10 mM PIPES-KOH, 10 mM MgCl2 6H2O, 2% PVP K40, 200 mM L-lysine monohydrochloride, 6 mM EGTA, pH 6
| Reagent | Sigma Aldrich cat # | 1 L | |
| Mannitol | M4125-1KG | 91 g | |
| PIPES-KOH | P7643-100G | 3.78 g | |
| MgCl2 6H2O | M9272-100G | 2.03 g | |
| PVP K40 | PVP40-500G | 20 g | |
| L-lysine monohydrochloride | L5626-1KG | 36.52 g | |
| EGTA | E3889-100G | 2.28 g |
Heat up 800 mL of deionized water, 2-4 min in microwave. Pour into a glass beaker with a magnetic stirrer. Add PVP K40, three spoonfuls at a time, stir vigorously. To speed up this process you can use two flat spatulas and “cut” the PVP blobs as if it was a steak.
Add mannitol and dissolve completely before adding PIPES, magnesium, lysine, and EGTA. Adjust the pH to 6.0 with 10 M NaOH. Adjust the volume to 1 L. Split the buffer into two 1 L Schott bottles. Autoclave and cool down to room temperature. Store at 4°C until ready to use.
- NEB complete and NEB –no b-mercaptoethanol
Just before starting the nuclei isolation add 0.9 g sodium meta-bisulphite (Sigma Aldrich cat # 255556-100G) to each Schott bottle. Close the bottle and shake until it is completely dissolved. The final concentration is 10 mM.
Add 0.2 mL β-mercaptoethanol to one Schott bottle only. Label this one as NEB complete buffer. The other flask is NEB – no β-mercaptoethanol. Keep on ice.
- 25% Triton X-100 in NEB complete
Transfer 7.5 mL of NEB complete buffer to a 50 mL Falcon tube. Add 2.5 mL of Triton X-100, use a wide bore tip. Aspirate the detergent very slowly from the stock bottle until you don’t see any change in the liquid level. Do not dip the tip too far into the stock bottle to avoid carrying extra detergent into the Falcon tube. Dispense the detergent into the 7.5 mL of buffer, do it very slowly. Aspirate some buffer to clean up the inside of the wide bore tip until there is no more detergent lumps visible. Close the tube and vortex thoroughly until fully dissolved. Spin down ~ 10 min at 3000 rpm at room temperature to pop all the bubbles. Keep the tube on ice.
- Tris-borate/K-acetate/SDS Lysis buffer: 0.5% SDS, 5 mM EDTA, 150 mM Tris-borate pH 7.4
To prepare this solution the Tris buffer is titrated with boric acid, instead of the usual HCl. Autoclave and keep at room temperature.
- CTAB/NaCl lysis buffer: 2% CTAB (hexadecyltrimethyl ammonium bromide), 2% PVP K40 (polyvinyl pyrrolidinone K40), 2 M NaCl, 25 mM EDTA, 100 mM Tris-HCl pH 8.0. To dissovle the PVP lumps, use the same technique as described above for NEB. Split the buffer into two 1 L Schott bottles and autoclave. Keep at room temperature.
- 5 M potassium acetate pH 7.0. Prepare according to Green and Sambrook 2012 and sterilize by filtration.
- 100% Ethanol
- 70% Ethanol
- Isopropanol
- Chloroform:isoamyl alcohol (24:1)
- β-mercaptoethanol (Sigma Aldrich cat # 63689)
- TE pH 7.5 (10 mM Tris-HCl, 1 mM EDTA pH 7.5)
- RNase A 100 mg/mL (Qiagen cat # 19101)
- Proteinase K 20 mg/mL (Qiagen cat # 19133)
- Qiagen DNeasy Plant Maxi kit (Qiagen cat # 68163)
- SybrSafe (Invitrogen cat # S33102)
- Lambda DNA (Invitrogen cat # SD0011)
- 1 kb+ ladder (Invitrogen cat # 10787018)
- Lambda ladder (BioRad cat # 170-3635)
- Standard agarose (Invitrogen cat # 16500500)
- PFGE grade agarose (BioRad cat # 1613108)
- 1X TAE gel running buffer
Equipment
- Waring commercial blender or a standard kitchen blender (for example, Sunbeam model PB7950)
- Miracloth
- Cheese cloth, sterile
- Two Large funnels
- Two 500 mL cylinder
- Parafilm
- Wide bore tips: 200, 1000 and 5000 μL, sterile, to pipette the purified nuclear genomic DNA and the Triton X-100 stock solution
- Kimwipes
- Qubit system with HS dsDNA kit or DeNovix (DS-11 FX, FX+ or QFX) with dsDNA High Sensitivity kit
- Microvolume spectrophotometer Nanodrop or DeNovix (DS-11, 11+, FX or FX+)
- Sorvall centrifuge or similar with SLA 600 TC rotor or SS34 rotor
- Nalgene centrifuge tubes Nalgene™ Oak Ridge (cat # 3119-0050)
- Bench top centrifuge with plate rotor (for example, Eppendorf SKU 5810000084 Rotor A-4-62, with 4 MTP buckets; Cat. # 5810711002)
- Falcon tubes, sterile, 50 mL
- Microcentrifuge
- 1.5 mL microcentrifuge tubes, sterile
- Gel electrophoresis box and power supply
- CHEF-DR III variable angle system for PFGE analysis (BioRad cat # 1703702)
- Water bath with shaker
Acknowledgements
The method has been improved thanks to my colleagues' interest in learning it: Paul Datson, Don Hunter, Mark Livermore, Elena Lopez Girona, John McCallum, Fatima Naim, Craig Woods, Melinda Zhang, and Qiong Zhang.
Nuclei isolation
Nuclei isolation
Pour 300 mL ice-cold NEB complete buffer in a blender. Work inside a fume hood.
Weight 20 g of kiwifruit leaves, cut any long stems and transfer to the blender
Homogenize for 30 sec in low setting
Note
(Note: If your blender is powerful, use low setting, but if using a kitchen blender set it on High)
Note
Do not use a stick hand-held blender because the sample won't be homogenized all at the same time.
Filtrate homogenate through 4 layers of cheesecloth into a sterile glass cylinder
Note
It is best to use sterile glassware, but if you are confident your washing techniques are thorough, use non-sterile glassware.
Note
If you can't find good quality cheese cloth, you can use disposable kitchen cloth (for example CHUX superwipes)
Filter again through 4 layers of miracloth into another 500 mL sterile glass cylinder
Note
Stack the miracloth layers against the fabric grain, i.e., perpendicular to each other
Adjust the volume to 294 mL with NEB complete buffer
Add 6 mL of 25% Triton X-100 in NEB complete buffer by placing the tip of the pipette on the inside wall of the cylinder, close to the top. Dispense the solution slowly and rotate the cylinder with your other hand. You will see the syrup-like solution smear downwards towards the homogenate.
Seal cylinder with parafilm and mix very gently by inversion 10-20 times. Rotate the cylinder every time you mix it
Aliquot the homogenate into six 50 mL Falcon tubes
Note
You can also use Nalgene centrifuge tubes. And instead of the SLA-600 TC rotor you will need the SS34
Spin down at 588 rpm (57 g) using the SLA-600 TC rotor at 4-10°C, for 2 min only. A tiny pellet might be visible. If no pellet is observed, continue with next step without changing tubes.
Note
If using the SS34, adjust the speed to correspond to the same g values used on the SLA-600 TC
00:02:00 Low speed centrifugation
Transfer the supernatant to a new set of 50 mL Falcon tubes and spin down at 3308 rpm (1800 g), same rotor, 10 °C, 15 min
00:15:00 Nuclei pellet centrifugation
10 °C Centrifugation temperature
Note
The centrifugal force depends on the genome size but also on the medium's density. The osmoticum used in this buffer (mannitol) is at 0.5 M and seems to work well with genomes upto 3 Gb (N. benthamiana), at 1800 x g, but please see recommendations here. Other cellular material such as starch might contribue to the easy sedimentation of large genomes such as the N. benthamiana.
Transfer supernatant to a waste container for proper disposal later.
Safety information
Check the waste disposal regulations for b-mercaptoethanol solutions and solid waste
Resuspend each pellet with 50 mL NEB –no β-mercaptoethanol. Mix gently by inversion until the pellet is resuspended. Spin down again as before.
go to step #10 Nuclei pellet centrifugation
Transfer supernatant to the waste container for proper disposal
Safety information
This supernatant does not contain β-mercaptoethanol and could be poured down the drain unless you are working with regulated biological material
Resuspend each pellet with 5 mL NEB –no β-mercaptoethanol and collect all pellets into one Falcon tube previously weighted. Add more NEB –no β-mercaptoethanol to final volume of 50 mL
Collect the nuclei by centrifugation
go to step #10 Nuclei pellet centrifugation
Weight the tube to find out the total amount of nuclei isolated. From 20 g of kiwifruit fully developed leaves expect about 0.2 g of nuclei, but this will vary depending on the species and genotype. Keep the tube on ice
Note
If you can’t continue with the nuclear genomic DNA extraction, store the nuclei pellet at -80°C until ready to extract the DNA
DNA extraction options
DNA extraction options
Depending on the nature of the tissue and the budget, select one of the following methods to isolate the nuclear genomic DNA.
DNA extraction I Tris-borate/K-acetate/SDS
DNA extraction I Tris-borate/K-acetate/SDS
Resuspend 0.2 g of nuclei pellet in 14 mL lysis buffer, mix by inversion, do not vortex
Note
A modified version of the method published by Lopez-Gomez and Gomez-Lim (1992) HortScience 27(5):440-442.
This protocol is suitable for plants with a high carbohydrate content.
Add 7 μL of RNase A 100 mg/mL. Mix by inversion
Incubate 10 min at 37°C with gentle shaking
37 °C RNA digestion
00:10:00 Lysis and RNA digestion
Add 1.4 mL 5 M K-Acetate pH 7, mix by inversion
Add 3.5 mL 100% Ethanol, vortex at maximum speed for 30 s
Extract the sample with an equal volume of chloroform:isoamyl alcohol (24:1). Mix by inversion 10 times, then place the tube horizontally in orbital shaker at room temperature and shake for 5 min, gently
00:05:00 Chloroform extraction
20 °C Chlorform extraction
Spin down at 3000 rpm 10 min at room temperature using a swing bucket rotor
00:10:00 Chloroform extraction centrifugaion
Transfer the aqueous phase to a new 50 mL Falcon tube. Add an equal volume of ice cold isopropanol. Mix by inversion and store at -20°C for 30 min
00:30:00 Isopropanol precipitation
-20 °C Isopropanol precipitation
Note
If the aqueous phase is not clear, repeat the chloroform extraction (steps 23-24)
Spin down at 3000 rpm for 25 min at room temperature or 10°C
00:25:00 DNA precipitation centrifugation
Discard supernatant and add same volume of 70% ethanol to wash the pellet. Let it stand at room temperature for ~ 10 min
00:10:00 Elute salts from DNA pellet
20 °C Elute salts from DNA pellet
Collect DNA by centrifugation
go to step #26 Centrifuge DNA pellet
Discard the supernatant. Remove all traces of ethanol with a kimwipe without touching the pellet.
Air dry the DNA pellet at room temperature for 20-30 min and add 100-200 μL TE buffer pH 7.5. Tap the bottom of the tube to detach the pellet and let it diffuse slowly at room temperature for a few hours or in the refrigerator overnight. Avoid vortexing the DNA solution at high speed.
Note
If you are concerned the DNA will bind to the tube irreversible, add the TE buffer immediately and leave the tube open, at room temperature. Any traces of ethanol will evaporte from the solution.
Store the DNA at 4°C. For long term storage, make numbered aliquots and store at -20°C. Use DNA aliquots sequentially and avoid freeze/thaw cycles
DNA Extraction II CTAB/NaCl/Proteinase K protocol
DNA Extraction II CTAB/NaCl/Proteinase K protocol
Add 3.5 mL of CTAB/NaCl extraction buffer and 80 μL Proteinase K to the nuclei pellet and vortex gently to resuspend
Incubate at 56°C for 1-2 hour without shaking or until all solution is clear
Add 10 μL RNase A 100 mg/mL, mix and incubate at room temperature for 5 min
00:05:00 RNase A digestion
Add 3.5 mL of chloroform:isoamyl alcohol (24:1) and vortex until an emulsion is obtained
Separate the phases by centrifugation using a swing bucket rotor, 3000 rpm, at room temperature for 15 min
00:15:00 Chloroform extraction centrifugation
If the interphase between the organic and aqueous solutions is not tight, you could centrigue the sample for another 15 min or transfer the aqueous phase to a new tube and add equal volume of chloroform:isoamyl alcohol
go to step #34 Chloroform extraction
go to step #35 Chloroform extraction centrifugation
Transfer aqueous phase to a new Falcon tube and add 2 volumes of 100% ethanol. Mix gently. Incubate at -20°C for at least 2 h
02:00:00 Etahnol precipitation
-20 °C Ethanol precipitation
Collect the DNA by centrifugation as explained before (Step 26-30)
DNA extraction III Qiagen DNeasy Plant Maxi Kit
DNA extraction III Qiagen DNeasy Plant Maxi Kit
Follow the Qiagen DNease Plant Maxi kit instructions with the following modifications:
- The final pellet of nuclei should be divided into 2 tubes and treat each one as the starting material for one extraction using this kit
- To elute the DNA incubate the binding column with 1 mL of AE buffer for 1 h. Spin down as described in the kit instructions. Add another mL of AE buffer to the binding column and incubate at room temperature for 30 min. Spin down again. Each column/tube will have ~ 2 mL of eluted DNA
- Combine both samples (~ 4 mL) and add 1/10 volume of 3 M sodium acetate pH 5.2, mix, and add 2 volumes of 100% ethanol. Mix. Store at 4°C or -20°C ovenight
- Collect, wash and resuspend the pellet as described in steps 26-30
The fragment size distribution obtained with this kit is 20-40 kbp
DNA quality check
DNA quality check
Check the quality of the ngDNA by absorbance ratios at 260/280 nm and 260/230 nm. Aim for ~1.8 and >2.0, respectively.
Quantify the ngDNA by fluorescence (Qubit or DeNovix) using a high sensitivity kit. Make 10-fold serial dilutions and quantify 1-5 μL
Run 100- 200 ng of ngDNA in a standard 1% agarose gel, stained with Sybr safe. Use lambda DNA (48.5 kbp) as control.
If you have access to a pulse field gel electrophoresis system, run ~ 200 ng against a lambda ladder, intact lambda DNA and 1 kb+ ladder.
Expected result