TITLE: eDNA extraction from water samples filtered through 47 mm diameter filters  (NucleoMag DNA/RNA Water Kit - MACHEREY NAGEL).
AUTHORS: Marine Vautier, Cecile Chardon, Cyrielle Galiegue, Isabelle Domaizon

[DESCRIPTION]
The objective of this protocol is the environmental DNA (eDNA) extraction from water samples filtered through 47 mm diameter filters. This protocol can be performed from filters preserved in tubes prefilled or not with preservation buffer.

DNA extraction is performed using a MagnetaPure 32 Nucleic Acid Purification System (Dutscher) and with the NucleoMagDNA/RNA Water Kit (Macherey Nagel). 

The procedure is based on reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. 

The benefits of using the MagnetaPure 32 Nucleic Acid Purification System (Dutscher) are increased productivity and repeatability, as well as eliminating human error and the pain of repetitive work.

This protocol is used prior to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target both macro- and micro-organisms eDNA extracted from water samples.

This protocol is optimised for rare eDNA and the suggested elution volumes are therefore low (between 50 and 65 µL), but can be increased if targets are more abundant.

[BEFORE_START]
Filtration and preservation of the water samples through filters

This protocol can be performed from filters preserved in tubes prefilled or not with preservation buffer (e.g. MWA1). If the filter is not prefilled with buffer, it should be frozen immediately after water filtration and until DNA extraction. 

Note: If the filters are preserved with MWA1 buffer, the extraction can be performed directly, but for other preservation buffers such as Longmire and CTAB, the protocol proposed here will work, but upstream precipitation is recommended to increase the extraction efficiency.

This protocole is suitable for the DNA extraction from different types of 47 mm diameter filters:  
 


The filters should be preserved in 5 mL tubes for this DNA extraction protocol.


The following precautions must be applied:

- Wear gloves throughout the extraction process
- Clean the bench with a DNA-removing solution (e.g. DNA-off, DNA away).
- 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)

For any manipulation in a rare DNA room, provide complete equipment (disposable coat, cap, mask, shoe covers & gloves).

Pre and post extraction equipment decontamination:

      - Clean a specific DNA work station and apply UV
      - MagnetaPure 32 System (Dutscher): Visual check for residues to be removed and UV decontamination – see instrument manual 

Scissors decontamination (to be done before starting the protocol, and between each filter cutting)

- Prepare : 
one 50 mL tube with DNA-removing solution (e.g. DNA-off, DNA away)
one 50 mL tube with molecular biology grade water
one 50 mL tube with ethanol
- Successively dip the scissors into each tube, opening and closing the scissors in each tube.

Note: The cutting of filters is not a requirement, but it does have an impact on the extraction efficiency.


[GUIDELINES]
The main steps of the protocol are:

Material preparation
Plate preparation
Sample lysis
Extraction with the MagnetaPure 32 System (Dutscher)
DNA elution

[STEPS]
SECTION: Material preparation
1. 
To limit contamination of the kit buffers, it is recommended to aliquote them:
- Into 50 mL tubes for MWA1, MWA2, MWA3 and MWA4
- Into 2 mL tubes for NucleoMag B-Beads solution and DNase-free H2O

Tubes annotation 
- one 2 mL tube per sample for lysate collection
- one 1.5 mL tube per sample for DNA collection
- one 5 mL tube per 8 samples for the NucleoMag B-Beads and MWA2 mix preparation

Scissors decontamination (to be done before starting the protocole, and between each filter cutting)
- Prepare : 
one 50 mL tube with DNA-removing solution (e.g. DNA-off, DNA away)
one 50 mL tube with molecular biology grade water
one 50 mL tube with ethanol
- Successively dip the scissors into each tube, opening and closing the scissors in each tube.

Note: The cutting of filters is not a requirement, but it does have an impact on the extraction efficiency.
SECTION: Plate preparation 1/2
3. Preparation of the NucleoMag B-Beads and MWA2 mix:
- Prepare one 5 mL tube / maximum 8 samples  
- Add MWA2 only, the NucleoMag B-beads will be added during plate preparation 2/2 (allow a margin of one sample for the mix preparation. For example: plan a mix for 9 samples if 8 samples are to be extracted)

 

SECTION: Plate preparation 1/2
2. In this step, the buffers provided by the kit are distributed in a 96-well plate.
For DNA extraction from filtered water samples, the 12 columns of the plate are divided into 2 sections of 6 columns each, allowing up to 16 samples to be extracted per plate.

Annotate the 96-well plate as recommended below:
 

Note: It is useful to mark the dividing line between columns 6 and 7 with a marker pen to provide a visual cue for filling the plate.

Add the appropriate buffers into the appropriate wells of the plate.

1stcolumn / 7thcolumn: Will be filled during plate preparation 2/2
2ndcolumn / 8thcolumn:   850 µL of MWA3 (1st wash)
3rdcolumn / 9thcolumn:   850 µL of MWA3 (2nd wash)
4thcolumn /10thcolumn:   850 µL of MWA4  (3rd wash and bead drying)
5thcolumn / 11thcolumn: not used
6thcolumn / 12thcolumn:   50 µL or 65 µL of DNase-free  H2O (DNA elution)

Note: The choice of elution volume is based on the expected eDNA amount. The smaller the amount, the smaller the elution volume in order to obtain more concentrated DNA.

 

Film and reserve the plate at Room temperature
SECTION: Sample Lysis
4. During this step, a mechanical and chemical lysis of the sample is performed.

For samples prefilled with buffer:
- Collect the 5 mL tubes containing the filters 
Note: If the tubes containing the filters are frozen, defrost them 10 min atRoom temperature
- Cut the filter into small pieces directly into the tube using decontaminated scissors
Note: Scissors must be decontaminated between each sample (see section 1 "material preparation")
- Place the tubes on the vertical vortex 5 s at median speed
- Place the tubes on the horizontal vortex 5 s at maximum speed
- Place the tubes into the benchtop centrifuge
- Pipette the lysate from the tube and transfer it into a 2 mL tube
- Centrifuge at   
- Replace the tubes into the rack and reserve them at Room temperature until their distribution into the 96-well plate

For samples without buffer:
- Collect the 5mL tubes containing the filters from the freezer and place on ice 
- Add immediately 750 µL of MWA1 buffer into each tube
- Cut the filter into small pieces directly into each tube using decontaminated scissors
Note: Scissors must be decontaminated between each sample (see section 1 "material preparation")
- Place the tubes on the vertical vortex 5 s at median speed
- Place the tubes on the horizontal vortex 5 s at maximum speed
- Place the 5 mL tubes into the benchtop centrifuge
- Pipette the lysate from the tube (approximately 650 µL) and transfer it into a 2 mL tube
- Centrifuge at   
- Replace the tubes into the rack and reserve them at Room temperature until their distribution into the 96-well plate

SECTION: Plate preparation 2/2
5. Preparation of the NucleoMag B-Beads and MWA2 mix 
NucleoMag B-Beads and MWA2 mix sediment quickly, vortex between each samples to ensure homogeneity     

- Vigorously vortex NucleoMag B-Beads tube 
- For each tube containing MWA2 buffer (previously prepared), add the appropriate volume of NucleoMag B-Beads (see Table 3 above)  
- Vortex

NucleoMag B-Beads - MWA2 mix and lysate distribution
- Remove the film from the plate
- Add the appropriate solution into each well of the plate 

1stcolumn / 7thcolumn: 
500 µL of NucleoMag B-Beads and MWA2 mix 
450 µL of Lysate (supernatant from the 2 mL tubes)

 

SECTION: Extraction step performed in the MagnetaPure 32 System
6. Place the plate into the MagnetaPure 32 System and insert the magnetic rod coverfor – see instrument manual

- Select the appropriate program to the chosen elution volume and elution temperature
 

Note: Heating to 56°C during elution gives a higher yield of DNA, but there is a risk of evaporation which reduces the volume of eluted DNA recovered

- Start the run (The run lasts approximately 40 min)

SECTION: Transfer of DNA extracts
7. At the end of the run, remove the plate and place it into the DNA-workstation
- Remove the magnetic rod cover and start UV for decontamination – see instrument manual
- In the DNA-workstation, transfer each DNA extract into a 1.5 mL tube previously annotated

Note:  DNA concentration and quality can be measured at this step (e.g. Nanodrop)

Store DNA extracts at 4 °C for immediate use, or at -20 °C or -80 °C for long-term preservation