Mar 19, 2025
Purification of marine DNA virus by sucrose density gradient.
- 1Station biologique de Roscoff, UMR7144 CNRS Sorbonne Université
- Ecology of Marine Plankton (ECOMAP) team - Roscoff
- AD2M
Protocol Citation: Pauline nogaret, Anne-Claire Baudoux, Estelle Bigeard 2025. Purification of marine DNA virus by sucrose density gradient.. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1zwdlmk/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: July 09, 2024
Last Modified: March 19, 2025
Protocol Integer ID: 103062
Keywords: virus, extraction, gradient sucrose, purification, DNA virus, nucleic acid extraction, marine ecology, environmental samples, DNA viruses
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-22-CE01-0018
Abstract
This protocol describes our workflow for processing the virus lysate to study the genome of marine DNA viruses. The steps include lysate clarification, viral concentration and purification using sucrose density gradient, DNA extraction and a last step of nucleic acids quantification. The DNA obtained using this protocol can be use for sequencing.
This method was adapted to F.Lelchat et al.,2019.
Image Attribution
Transmission electron micrograph of the marine bacterium Cobetia marina DSMZ 4741 infected by an Carin5 bacteriophage (Lelchat F et al. 2019).
Guidelines
Linear sucrose gradient tube preparation :
Materials
Equipments :
White fluorescent light (Philips Master, cat no : TL_D 18W/865)
Micropipets (Eppendorf)
Hand pump
Centriguge Avanti J-25 XP & JA10 Rotor (Bekman Coulter)
Cork SW40/41 x6 (Science Services GmbH, cat no : 105-414-6)
Cannula in inox autoclaved
Consumables :
1L Flasks with Vented Filter Caps (Starlab, cat no : CC7682-4822)
500mL autoclaved centrifuge bottle (VWR, cat no : 525-2313)
Tubes 1,5mL 2mL 15mL
Pipet 10ml (Dominique Dutscher, cat no : 607180)
Filter tips
Filtration unit 0,45µm, Durapore PVDF membrane, 500 mL (Merck Millipore, cat no : S2HVU05RE)
Filtration unit 0,22µm, Durapore PVDF membrane, 500 mL (Merck Millipore, cat no : S2GVU05RE)
Vivaflow 200 30kDa, MWCO, PES (Sartorius, cat no : VF20P2) - stored in ethanol at 4°C
Vivaspin 20 30kDa, MWCO, PES (Cytiva, cat no : 28932361)
Open-Top Thinwall Ultra-Clear Tube, 14x89mm, 13,2mL (50Pk) (Beckman Coulter, cat no : 344059)
Sterile needle 21-25G (Dominique Dutscher, cat no : 050139B)
Sterile syringe 3mL (Dominique Duscther, cat no : 050829)
Syringe Filter 0,2µm 25mm,PES (Fisher scientific, cat no : 15392388)
Adhesive (Scotch)
Lamp
Reagent:
NaCl (Merck Sigma, cat no : S9888)
MgSO4. 7H20 (Merck Sigma, cat no : 63138)
Tris-HCl (Merck Sigma, cat no : T5941)
Sucrose (Merck Sigma, cat no : S0389-1KG)
Qubit dsDNA BR Assay Kit (Invitrogen, cat no : Q32853)
DNeasy Blood and Tissue Kit (Qiagen, cat no : 69504)
Absolute ethanol (Fisher scientific, cat no : 10342652)
Ice
Before start
Preparation of solutions:
1M Tris HCl pH 7.5:
15.76g Tris HCl + MilliQ water (qsp 100mL) and adjust pH at 7,5.
SM Buffer (autoclaved and filtered through a 0,2µm filter):
5,8g NaCl (100mM) + 2g MgSO4.7H20 (8mM) + 50mL 1M Tris HCl pH 7,5 (50mM) + MilliQ water (qsp 1L).
Sucrose 10% : 10g of sucrose fill up to 100mL of SM buffer.
Sucrose 40% : 40g of sucrose fill up to 100mL of SM buffer.
10% and 40% sucrose solution are sterilized either by filtration through a 0.2 µm PES syringe filter or by autoclaving at 121°C for 15 minutes.
Lysate preparation.
Lysate preparation.
3w
3w
500mL of marine viruses cuture was grown in the presence of its host in appropriate medium at 18°C, under a 12:12 h light:dark cycle of 100 μmol photons·m⁻²·s⁻¹ provided by a white fluorescent light.
Viral clarification and concentration.
Viral clarification and concentration.
1h
1h
Lysate clarification to remove cellular debris :
- Centrifuge the viral lysate (500mL) 5000 rcf, 4°C, 00:30:00 (Avanti J-25XP, Beckman Coulter-JA10 Rotor).
- Carefully transfer the supernatant without disturbing the pellet into a 0.45µm Durapore filtration unit and filter thoroughly using a hand pump. It's possible to refilter on 0,2µm filtration unit to remove more debris.
30m
Viral concentration :
- Ultrafilter the filtrate using a Vivaflow 200 30kDa PES cartridge to concentrate the sample down to a volume of 50mL.
- Further concentrate the sample using a Vivaspin 20 30kDa PES centrifugal concentrator 6000 x g, 4°C, 00:10:00 (Eppendorf, 5804-R).
- Repeat this step until the volume is reduced to 1mL. Ensure that the filtrate is removed after each step to prevent overflow.
- Store the Vivaspin at 4°C overnight to improve virus recovery.
- Transfer the concentrate in a 2mL microtube.
- Rinse the membrane with 1 mL of appropriate buffer or ultrafiltrate and transfer the rinse in the 2mL microtube.
1d 5h
Viral purification using linear sucrose gradient.
Viral purification using linear sucrose gradient.
4h
4h
Gradient tube preparation :
Prepare two sucrose gradients per sample.
Freshly prepare 10% (w/v) and 40% (w/v) sucrose solutions. Sterilize either by filtration through a 0,2 µm PES syringe filter or by autoclaving at 121°C for 15 minutes.
Gradients are prepared in open-top thinwall Ultra-Clear tubes using a Master Gradient Maker.
- Label each ultracentrifuge tube and mark the half-full level (approx. 7 mL) using the Master Gradient marker block.
- Using a sterile disposable pipette, fill all tubes with air-free, room temperature light solution (10% sucrose solution) above the half-full mark.
- Attach the cannula to a sterile syringe.
- Fill the syringe with air-free, room temperature heavy solution (40% sucrose solution) and insert the cannula quickly to the bottom of the tube and begin layering the heavy solution (see Figure 1). Holding the tube at eye level, carefully fill until the heavy-light interface rises precisely to the half-full mark. Avoid disturbing the light solution.
- Withdraw the syringe quickly and smoothly.
- Add or remove light solution so that there is enough to fill air hole when the cap is inserted (see Figure 1)
- Level and calibrate the Gradient Master‱ machine according to the manufacturer’s recommendations.
- Place the tubes into a magnetic tube holder (MagnaBase‱) and position it on the instrument.
- Generate the gradient using the 10%-40% sucrose gradient program.
Figure 1 : Realization of sucrose gradient.
1h
Particle separation:
- Gently add 400-450µL of the viral concentrate on the top of the sucrose gradient.
- Weight and balance the tubes.
Figure 2 : Adding of viral concentrated solution.
- Utracentrifuge (Optima XPN-80, Beckman, Sw41Ti Rotor) :
134400 x g, 4°C, 00:30:00 for cyanophages.
68600 x g, 4°C, 00:30:00 for micro-algae viruses.
Collection of the purified virus suspension :
- ln a dark environment, illuminate the tube from above with a lamp to visualize the white band containing the viruses.
- Using a 21G needle and a 1 mL syringe, carefully pierce the tube and collect the white band.
- Transfer the purified viral suspension into a sterile tube. If multiple tubes originate from the same sample, the collected bands can be pooled.
Figure 3 : Visualization of virus white bande.
2h
Bands cleaning :
- Transfer the purified viral suspension in the concentrate pocket of a Vivaspin 20 30 kDa PES centrifugal concentrator and fill up to 15 ml with SM buffer.
- Centrifuge 6000 x g, 4°C, 00:10:00 (Eppendorf, 5804-R).
- Discard the eluate and resuspend the retentate in 10mL of SM buffer.
- Centrifuge 6000 x g, 4°C, 00:10:00 (Eppendorf, 5804-R).
- Repeat this cleaning step 2 times to remove traces of sucrose.
- Store the viral suspension in the Vivaspin at 4°C overnight.
- Collect the purified viral solution (1mL).
1d 1h
Extraction of DNA.
Extraction of DNA.
Purification of total DNA :
Use the animal Blood or Cells (Spin column protocol) of DNeasy Blood and Tissue Kit (Qiagen).
Dispense 200µL of the purified viral suspension in a clean microtube (tube 1). Use as many tubes as necessary to extract the whole virus suspension volume (tube 2, ....).
For each tube :
- Add 20μL Proteinase K.
- Add 200μL Buffer AL (without added ethanol). Vortex and incubate at 56°C for 1 hour.
- Add 200μL ethanol at RT (100%) to the sample, and mix by vortexing.
- Pipette the mixture into the DNeasy Mini spin column placed in a 2mL collection tube.
- Centrifuge at 6800 g for 1 min 12°C (Eppendorf, 5427-R) . Discard flow-through and collection tube.
- Place the column in a new 2mL collection tube, add 500μL Buffer AW1.
- Centrifuge for 1 min at 6000 g 12°C(Eppendorf, 5427-R). Discard flow-through and collection tube.
- Place the column in a new 2mL collection tube, add 500μL Buffer AW2.
- Centrifuge 30s at 6000 g 12°C (Eppendorf, 5427-R). Discard flow-through and tube.
- Change collection tube and centrifuge for 1 min at 20,000 g 12°C. Column need to by dry.
- Place the DNeasy Mini spin column in a tube 1.5mL and pipette 100μL Buffer AE directly onto the DNeasy membrane for only tube 1.
- Incubate at room temperature for 1 min.
- Centrifuge for 1 min at 6800 g 12°C to elute DNA (Eppendorf, 5427-R).
- Transfer 100μL of eluate-tube 1 on the column “tube1”.
- Centrifuge for 1 min at 6800 g 12°C to elute DNA (Eppendorf, 5427-R).
- Transfer 100μL of eluate-tube 1 on the column “tube2”. DNA from tube 1 and tube 2 will be pooled.
- Centrifuge for 1 min at 6800 g 12°C to elute DNA (Eppendorf, 5427-R).
- Transfer 100μL of eluate- tube 2 on the column “tube2”.
- Centrifuge for 1 min at 6800 g 12°C to elute DNA (Eppendorf, 5427-R).
- Repeat these steps with the other tubes in order to increase the DNA yield.
- Keep 2,5µL for QuBit quantification (place this aliquot at -20°C if the quantification is done later) and store the DNA sample at -20°C.
QuBit quantification using Qubit 4 fluorimeter.
QuBit quantification using Qubit 4 fluorimeter.
Quantify the DNA concentration using the Qubit 4 fluorimeter and the dsDNA BR Assay kit which provides a detection range of 4 and 2000ng of DNA.
A Agilent Bioanalyzer quantification is possible.
Protocol references
F Lelchat, P Y Mocaer, T Ojima, G Michel, G Sarthou, E Bucciarelli, S Cérantola, S Colliec-Jouault, C Boisset, A-C Baudoux, Viral degradation of marine bacterial exopolysaccharides, FEMS Microbiology Ecology, Volume 95, Issue 7, July 2019, fiz079, https://doi.org/10.1093/femsec/fiz079
Acknowledgements
The image of the Carin-1 virus was established by Guy Shoehn's team at the Institut de Biologie Structurale.