Apr 06, 2022
isolation and extraction of plant nuclei in plug
- 1Genoscope/CEA
Protocol Citation: Karine Labadie, Benoît Vacherie 2022. isolation and extraction of plant nuclei in plug. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr6632vmk/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: February 03, 2022
Last Modified: April 06, 2022
Protocol Integer ID: 57758
Keywords: dna extraction, HMW, UHMW, Plug, optic card, bionano, extraction, plants, nuclei,
Abstract
Method for isolation and extraction of plant cell nuclei.
Protocol for obtaining UHMW DNA (> 150kb) allowing the production of optical cards with Bionano technology.
Guidelines
Never use a vortex in order to maintain a good molecule size.
Use only wide bore tips.
Allow the DNA to resuspend for a minimum of 48 hours before proceeding with QC.
The nuclei isolation part should be done in a chemical hood because of the use of 2-mercaptoethanol
Materials
Reagents :
Tris HClP212121
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
KCl
SucroseP212121
PVPSigma
Spermine
Spermidine
Triton X-100Sigma AldrichCatalog #T8787-50ML
2-MercaptoethanolSigma Aldrich
N-lauryl sarcosineSigma AldrichCatalog #L5125-50G
NaCl
UltraPure™ Low Melting Point Agarose Catalog #16520-100
Agarase (0.5 U/µL)Thermo FisherCatalog #EO0461
Proteinase K
RNase A
Dialysis membrane, 44mmBio Basic Inc.Catalog #TX0112.SIZE.2m
Consumables :
MBP™ Wide Bore Pipette TipsThermo FisherCatalog #02707600
CHEF Disposable Plug MoldsBioRad SciencesCatalog ##1703713
Certified CheeseclothThermo FisherCatalog #22055053
MiraclothMerck MilliporeCatalog #475855
Falcon 40 µm Cell StrainerCorningCatalog #352340
Petri DishP212121Catalog #LI-PD01100
Nunc™ Cell Factory™ System Accessories, vent capThermo FisherCatalog #146008
Equipment :
Equipment
ThermoMixer® C
NAME
Eppendorf
BRAND
Catalog No. 2231000680
SKU
LINK
Equipment
C1 Platform Shaker
NAME
Platform Shaker
TYPE
New Brunswick Scientific
BRAND
SKU unknown
SKU
Protocol materials
Nunc™ Cell Factory™ System Accessories, vent capThermo FisherCatalog #146008
Materials, Step 30
N-lauryl sarcosineMerck MilliporeSigma (Sigma-Aldrich)Catalog #L5125-50G
Materials
MBP™ Wide Bore Pipette TipsThermo FisherCatalog #02707600
Materials
RNase AQiagenCatalog #19101
Step 31
Proteinase K
Materials
SucroseP212121
Materials
2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)
Materials
Dialysis membrane, 44mmBio Basic Inc.Catalog #TX0112.SIZE.2m
Materials
NaCl
Materials
Agarase (0.5 U/µL)Thermo FisherCatalog #EO0461
Materials
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Materials
PVPMerck MilliporeSigma (Sigma-Aldrich)
Materials
RNase A
Materials
Certified CheeseclothThermo FisherCatalog #22055053
Materials, Step 10
MiraclothMerck Millipore (EMD Millipore)Catalog #475855
Materials, Step 10
Petri DishP212121Catalog #LI-PD01100
Materials
UltraPure™ Low Melting Point Agarose Catalog #16520-100
Materials, Step 20
CHEF Disposable Plug MoldsBio-Rad LaboratoriesCatalog ##1703713
Materials, Step 21
Falcon 40 µm Cell StrainerCorningCatalog #352340
Materials, Step 10
KCl
Materials
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Materials
Spermine
Materials
Spermidine
Materials
Tris HClP212121
Materials
preparation of reagents
preparation of reagents
NIB Buffer : 200 ml : freshly prepared
| Reagent | Final concentration | |
| Tris ph8 | 10 mM | |
| EDTA | 10 mM | |
| KCl | 80 mM | |
| Sucrose | 0.5 M | |
| PVP 40 | 2 % | |
| Spermine | 1 mM | |
| Spermidine | 1 mM | |
| H2O | qsp 200 ml |
Adjust the Ph to 9.4 then filter at 0.22 µm
NIBT Buffer : 160 ml
| A | B | |
| NIB Buffer | 160 ml | |
| Triton X100 | 0.5% |
NIBTM Buffer : 40 ml
| A | B | |
| NIBT Buffer | 40 ml | |
| 2-Mercaptoethanol | 0.75 % |
Cell suspension Buffer : Can be stored for 1 year at 4°C.
| A | B | |
| Tris ph8 | 10 mM | |
| EDTA | 50 mM | |
| NaCl | 2 mM | |
| H2O | Qsp 100ml |
Lysis Buffer : Can be stored for 1 year at RT.
| A | B | |
| EDTA 0.5M | 100 ml | |
| N-Lauroylsarcosine | 1 % |
Nuclei isolation
Nuclei isolation
3h
3h
Putting a mortar in ice
Cool a mortar/pestle with liquid nitrogen until the bubbling stops.
Place a beaker in ice and add a magnetic stirrer.
Add 20 ml of NIBTM (10 ml/g of leaves) and stir gently
20 mL NIBTM
Grind 2g of frozen sample for without adding liquid nitrogen, until a fine powder is obtained (approx. 2 min)
2 g 00:02:00
Before grinding
After grinding
2m
Transfer the powder to the beaker and shake gently for 10 minutes in ice.
00:10:00
10m
Filter the mixture into a 50ml tube through autoclaved filters (2 cheese cloth + 2 Mira cloth) on a funnel (squeeze the filters at the end of filtration to recover more of the solution containing the nuclei).
Filter through a 40µm cell stariner into a new 50ml tube.
Certified CheeseclothEmd MilliporeCatalog #22055053 MiraclothEmd MilliporeCatalog #475855
Falcon 40 µm Cell StrainerEmd MilliporeCatalog #352340
Pelleting the homogenate by centrifugation.(acceleration and deceleration at level 3)
800 x g, 4°C, 00:20:00 , Acc 3 / Dec 3
20m
Remove the supernatant and gently resuspend the pellet in ice (use a brush if the pellet does not recover).
Add 20ml of cold NIBTM
20 mL NIBTM
Pelleting the homogenate by centrifugation to remove residues and unlysed cells.
60 x g, 4°C, 00:02:00 , Acc 3 / Dec 3
2m
Filter the supernatant through a 40 µm cell sieve into a new 50 ml tube.
Pellet the nuclei by centrifugation.
800 x g, 4°C, 00:15:00 , Acc 3 / Dec 3
15m
Wash the pellet 3 times in NIBT buffer :
- Remove the supernatant
- Gently resuspend the pellet
- Add 40 ml of cold NIBT buffer 40 mL NIBT
- Centrifuge 800 x g, 4°C, 00:15:00 , Acc 3 / Dec 3
1st wash
2nd wash
3rd wash
15m
Make a final wash in 30 ml of cold NIB buffer.
800 x g, 4°C, 00:15:00 , Acc 3 / Dec 3
Final wash
15m
Resuspend the last pellet in the residual buffer (approx. 200 µl) and transfer the homogenate to a 1.5 ml tube.
Centrifuge.
800 x g, 4°C, 00:15:00 , Acc3 / Dec 3
15m
Remove the supernatant with a pipette and resuspend the pellet in an appropriate volume of cell suspension buffer :
60µl / plug
Adjust the number of plugs to be made according to the size of the pellet.
optional : microscopic observation
- Take an aliquot of 100 µl of suspension
- Stain with DAPI
- Observe the presence of nuclei
Embedding in agarose
Embedding in agarose
30m
30m
Use Low Melting agarose for a final agarose concentration of 0.8 %
UltraPure™ Low Melting Point Agarose Emd MilliporeCatalog #16520-100
Put a CHEF Disposable Plug Molds on ice
CHEF Disposable Plug MoldsEmd MilliporeCatalog ##1703713
Melt agarose at 70°C for 5 min then equilibrate at 43°C for 5 min
00:05:00 70 °C
00:05:00 43 °C
10m
Preheat the nuclei suspension to 43°C for 3min and then add the appropriate amount of 2% agarose (see table).
Mix gently with a wide-bore tip, avoiding bubbles.
00:03:00 43 °C
3m
Immediately dispense 100µl of mixture per well using wide-bore tips.
Allow to polymerise for 15 minutes on ice.
00:15:00 On ice
15m
Proteinase K digestion
Proteinase K digestion
18h
18h
Prepare a fresh proteinase K digestion solution by mixing 200 μl of proteinase K enzyme (20mg/ml) with 2.5 ml of lysis buffer in a 50 ml tube.
200 µL Prot. K 2.5 mL Lysis Buffer
Transfer plugs to the 50ml tube containing Proteinase K digestion solution.
Incubate in thermomixer for 2 hours at 50 °C with intermittent mixing
Mixing cycle: 10 seconds at 450 rpm followed by 10 minutes at 0 rpm
02:00:00 50 °C
2h
Screw a sieve caps onto the tube and empty the solution. Change the proteinase K Solution bath as before.
Incubate in thermomixer overnight at 50 °C with intermittent mixing
Mixing cycle: 10 seconds at 450 rpm followed by 10 minutes at 0 rpm
Overnight 50 °C
RNase Digestion
RNase Digestion
1h 30m
1h 30m
Prepare the wash solutions :
TE 10:50 (Wash Buffer)
TE 10:5 (For Rnase)
Empty the tube using a vent cap.
Nunc™ Cell Factory™ System Accessories, vent capEmd MilliporeCatalog #146008
Rinse the plugs 3 times with 10ml of wash buffer.
Wash 2 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a horizontal platform mixer.
15 rpm, Room temperature , 00:15:00
Rinse the plugs 3 times with 10ml of TE 10:5
Add 2.5ml of TE 10:5 and 50 µl of Rnase SolutionRNase AEmd MilliporeCatalog #19101
Incubate 1hour at 37°C with intermittent mixing
01:00:00 37 °C
1h
Rinse the plugs 3 times with 10ml of Wash Buffer.
NB : The plugs can be stored at 4°C in a wash buffer at this stage
Agarase treatment
Agarase treatment
2h
2h
Wash 4 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a horizontal platform mixer.
15 rpm, Room temperature , 00:15:00
Transfer the plug to a 1.5 ml tube with a sterile spatula
Melt the plug in a water bath at 70°C for 2 minutes
00:02:00 70 °C
2m
Transfer the tube to a water bath at 43°C for 5 minutes
00:05:00 43 °C
5m
Add 2µl of agarase and mix gently by rotating with the tip.
Incubate 45 minutes at 43 °C
00:45:00 43 °C
45m
Dialysis
Dialysis
1h
1h
Place 10 ml of 1x TE Buffer in a 6 cm Petri dish.
Float a 0.1 μm dialysis membrane on the surface of the 1x TE Buffer. Place a cover on the Petri dish and let the membrane equilibrates for 15 minutes.
00:15:00
15m
Deposit the entire sample in the centre of the membrane using a wide-bore tip.
Place cover on the Petri dish and let the sample dialyze for 45 minutes at room temperature.
00:45:00 Room temperature
45m
Transfer DNA to a 1.5 ml microfuge tube with a Wide Bore Tip.
Allow the DNA to resuspend overnight at RT then 2 days at 4°C before performing quality control.
Overnight Room temperature
Sample QC
Sample QC
Quantify your sample with a Qubit HS.
NB : Before quantification, sonicate the DNA aliquot for 10 min to obtain a more reliable result
Visualise 1 µL of sample to estimate the molecular weight. (Tapestation or/and pipin pulse or/and Femto pulse)
Résults
Résults
QC results obtained on different plant species, using different technologies to estimate the size of the molecules.