Apr 07, 2020
DNA extraction from environmental biofilm using the NucleoSpin® Soil kit (MACHEREY-NAGEL)
- Marine Vautier1,
- Valentin Vasselon2,
- Cecile Chardon3,
- Frédéric Rimet3,
- Agnès Bouchez3,
- Isabelle Domaizon1
- 1INRAE, CARRTEL, Pole R&D ECLA, Thonon les bains, France;
- 2Office Français pour la Biodiversité, Pole R&D ECLA, Thonon les bains, France;
- 3INRAE, CARRTEL, Thonon les bains, France
- EcoALpsWater
Protocol Citation: Marine Vautier, Valentin Vasselon, Cecile Chardon, Frédéric Rimet, Agnès Bouchez, Isabelle Domaizon 2020. DNA extraction from environmental biofilm using the NucleoSpin® Soil kit (MACHEREY-NAGEL) . protocols.io https://dx.doi.org/10.17504/protocols.io.bd52i88e
Manuscript citation:
Vasselon V., Domaizon, I., Rimet F., Kahlert, M., & Bouchez, A. (2017a). Application of high‐throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter? Freshwater Science, 36, 162–177 Vasselon V., A. Bouchez F. Rimet, S. Jacquet, R. Trobajo, M. Corniquel, K.Tapolczai & I. Domaizon (2017b) Avoiding quantification bias in metabarcoding: Application of a cell biovolume correction factor in diatom molecular biomonitoring. Methods in Ecology and Evolution, 9(4): 1060-1069. Vasselon V., Rimet F., Tapolczai K., & Bouchez A. (2018). Assessing ecological status with diatoms DNA metabarcoding: Scaling‐up on a WFD monitoring network (Mayotte Island, France). Ecological Indicators, 82, 1–12.
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: March 24, 2020
Last Modified: April 07, 2020
Protocol Integer ID: 34714
Keywords: DNA extraction, environmental biofilm, Diatoms, biomonitoring,
Abstract
This protocol is part of the DNA workflow applied in the Eco-AlpsWater Project.
The methodological step described here is the extraction of DNA, this is a critical step for obtaining relevant results since molecular inventories might be influenced by the DNA extraction method used.
The choice of the methodology for biofilms DNA extraction is based on previous studies and in particular on the work done by Vasselon et al. (2017) where the authors have tested 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA (from pure diatom cultures and biofilms samples from streams and lakes).
For the Eco-AlpsWater project, after being sampled in lakes or rivers, biofilms are stored in 50 mL tubes in ethanol at 4°C, and for a maximum of 3 months before DNA extraction (the extraction should preferably be done in the month following the sampling).
The DNA extraction protocol presented below has been used in several recent studies (e.g. Vasselon et al 2017ab, 2018) focussed on the application of diatoms metabarcoding; this extraction is based on a protocol adapted from the NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications for biofilm DNA extraction.
Attachments
Guidelines
- Sample preparation
- Cells lysis
- Contaminants elimination
- DNA fixation and washing
- DNA elution
Materials
- Samples
- environmental biofilm
- preserved in ethanol (final concentration > 70% of ethanol)
- volume needed = 2mL of the homogeneized sample
- Reagents
- NucleoSpin® Soil kit (MACHEREY-NAGEL)
- Ethanol (96 - 100%), molecular grade to prepare buffer SW2
- Materials
- specific DNA-work station (sterile area equipped with air filtration and UV systems)
- refrigerated microcentrifuge for 1.5 to 2 mL tubes (relative centrifugal force needed: 11,000 to 18,000 x g)
- horizontal vortexer with microtube holder
- water bath
- pipettes : 1000 µL - 100 µL
- 2 trash cans : 1 for liquid and 1 for solid
- Consumables
- tips with filter :
> 1000µL : 12 tips per samples
> 100µL : 1 tip per samples
- 2 mL sterile microcentrifuge tube : 2 per sample
(1 to collect solution after filter lysate - step 5 and 1 to elute DNA - step 10 )
- gloves
Safety warnings
The manufacturer advise to wear gloves and goggles and to flow the safety instructions for 2 reagents :
- SB coutains Guanidinium thiocyanate 45 - 60%,
CAS number : CAS 593-84-0
Signal word : Irritant
Hazard phrases : 302, 412
Precaution phrases : 264W, 273, 301+312, 330
- SW1 coutains Guanidine hydrochloride 36 - 50% and 2 - propanol 20 - 35%
CAS number : CAS 50-01-1, 67-63-0
Signal word : Irritant and Flammable
Hazard phrases : 226, 302, 319, 336
Precaution phrases : 210, 260D, 264W, 280sh, 301+312, 330
Before start
- The following precautions must be applied :
- Wear gloves throughout the extraction process
- Clean the bench with DNA off
- Use tips with filters to avoid contaminations
- All steps have to be performed under a specific DNA-work station (sterile area equipped with air filtration and UV systems).
- Materials preparation :
- Clean a specific DNA - work station and apply the UV for 15min
- Turn on the centrifuge - cool at +4°C
- Solutions preparation :
- Check lysis buffer SL1 for precipitated SDS. Dissolve any precipitate by incubating the buffer at 30-40°C for 10 min and shaking the bottle every 2 min.
- Check buffer SW2 - before the first utilisation, you need to add the indicate volume of ethanol (96 - 100%) to buffer SW2 concentrate and mark the label of the bottle to indicate that ethanol was added. This solution is stable at room temperature (18 - 25°C) for at least one year
- Incubate the elution buffer SE at +50°C
Prepare the sample
Prepare the sample
- Shake the 50mL falcon tube containing the biofilm / ethanol mixture by inverting the tube in order to obtain a homogeneous solution
- Take 2 mL of this homogeneized solution by pipetting twice 1 mL into a 2 mL sterile microcentrifuge tube and close the cap
Note : between each pipetting, aspirate/discharge to resuspend the biofilm particles that may have sedimented at the bottom of the tube
- Centrifuge 18000 x g, +4°C, 00:30:00 ,
- Discard the supernantant
- Add 700 µL of buffer SL1 to the biofilm pellet
Note : check SL1 buffer - dissolve any precipitate by incubating the buffer at 30-40°C for 10 min and shaking the bottle every 2 min
- Resuspend the biofilm pellet
- Transfer the biofilm/SL1 mixture into the NucleoSpin® Bead Tube Type A
Adjust lysis conditions
Adjust lysis conditions
- Add150 µL of Enhancer SX
- Close the cap
Sample lysis
Sample lysis
- Attach the NucleoSpin® Bead Tubes horizontally to a vortexer
Note : using a special adapter or alternatively tape to fix the tube
- Vortex the samples at full speed at Room temperature (18 - 25°C) for 00:05:00
Precipitate contaminants
Precipitate contaminants
- Centrifuge 11000 x g, 00:02:00
- Add 150 µL of buffer SL3 and vortex for 00:00:05
- Incubate at 4 °C in a fridge for 00:05:00
- Centrifuge 11000 x g, 00:01:00
Filter lysate
Filter lysate
- Place a NucleoSpin® Inhibitor Removal Column (red ring) in a Collection Tube (2mL, lid)
- Load up to 650 µL of clear supernatant (obtained at the step 4) onto the filter
- Centrifuge 11000 x g, 00:01:00
- Repeat the load and the centrifuge step as many time as there is still some supernatant from step 4 to be filtered
After each centrifugation, collect the filtered liquid in a clean tube : 1 single tube for all the filtration
- Discard the NucleoSpin® Inhibitor Removal Column
Note : if a pellet is visible after the centrifugation, transfer the clear supernantant to a new collection tube (not provided in the kit) to get ride of this pellet, and continue with the clear supernatant
Adjust binding conditions
Adjust binding conditions
- Add 250 µL of buffer SB
- Close the lid
- Vortex for 00:00:05 , make a brief centrifugation
Bind DNA
Bind DNA
- Place a NucleoSpin® Soil Column (green ring) in a collection Tube ( 2mL)
- Load 550 µL of sample onto the column
- Centrifuge 11000 x g, 00:01:00
- Discard the flow through and place the column back into the collection tube
- Load the remaining sample onto the column
- Centrifuge 11000 x g, 00:01:00
- Discard the flow through and place the column back into the collection tube
Wash and dry silica membrane
Wash and dry silica membrane
Note : the same collection tube is used throughout the entire washing procedure to reduce plastic waste
1st wash :
- Add 500 µL of buffer SB to the NucleoSpin® Soil Column
- Centrifuge 11000 x g, 00:00:30
- Discard the flow through and place the column back into the collection tube
2nd wash :
- Add 550 µL of buffer SW1 to the NucleoSpin® Soil Column
- Centrifuge 11000 x g, 00:00:30
- Discard the flow through and place the column back into the collection tube
3rd wash :
- Add 650 µL of buffer SW2 to the NucleoSpin® Soil Column
Note : verify that ethanol was added to buffer SW2 during the first utilisation
- Centrifuge 11000 x g, 00:00:30
- Discard the flow through and place the column back into the collection tube
4th wash :
- Add 650 µL of buffer SW2 to the NucleoSpin® Soil Column
- Centrifuge 11000 x g, 00:00:30
- Discard the flow through and place the column back into the collection tube
Dry silica membrane
Dry silica membrane
Centrifuge 11000 x g, 00:02:00
Note : if for any reason, the liquid in the collection tube has touched the NucleoSpin® Soil Column after drying step, discard flow through and centrifuge again
Elute DNA
Elute DNA
- Place the NucleoSpin® Soil Column into a new microcentrifuge tube (not provided in the kit)
- Add 30 µL of buffer SE previously heated 50 °C to the column
- Do not close the lid and incubate at Room temperature for 00:01:30
- Close the lid and centrifuge 11000 x g, 00:00:30
- Discard the NucleoSpin® Soil Column and keep the tube cointaining the DNA
- We recommend storing DNA frozen at -20°C until preparation of DNA library for HTS (or at -40°C to -80°C for longer storage)