TITLE: Total nucleic acid extraction - NucleoMag® DNA/RNA Water Kit (MACHEREY-NAGEL Inc.)
AUTHORS: Adélaïde Roguet, Melissa Schussman

[DESCRIPTION]
Total nucleic acid extraction from wastewater using Maxwell(R) HT Environmental TNA Kit, custom (Promega)

[BEFORE_START]
1. Clean the working area and all equipment: wipe down with 10% bleach and let dry. Wipe down with 70% ethanol and let dry. Then, wipe down using RNase AWAY and let dry. 

2. Prepare the 50% ethanol solution (it must be fresh!)

3. Prepare the 6 purification plates: 
    - Wash 1 plate: Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.
    - Wash 2 plate (same as plate Wash 1): Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.
    - Ethanol Wash plate: Add 450 μl of 50% ethanol to each well required for purification. 
    - Elution plate: Add 100 μl of 25 mM Tris-HCl (pH 8.0) to each well required for purification.
    - Tip plate: Place KingFisher 96-tip comb into an empty KingFisher 96-well plate. While opening the 96-tip comb plate, pay attention to not touch the tips.
    - Lysis and Bind plate: Add 35 μL of Resin to each well required for purification (vortex the bottle at max speed before use). Add 50 μl of Alkaline Protease Solution custom (APA) to each well required for purification. Add 250 μl of cell lysis solution (CLD) to each well required for purification. Add 400 μl of Isopropanol (100%) to each well required for purification.




[GUIDELINES]
When work is completed, remove equipment and supplies from the cabinet. Wipe the work area with 10% bleach, let stand for 10 min, rinse with water, then with 70% ethanol, and finally with RNAase AWAY.

[STEPS]
SECTION: Total nucleic acid extraction
1. For HA filter extraction, let the sample thaw on ice and go to step 2. 

For BCoV/BRSV extraction (in duplicate), add 5 µL of BCoV/BRSV solution to the 2-mL tube containing 475 µL MWA1. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.

For Direct extraction, add 150 µL of wastewater to the 2-mL tube containing 100 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.
SECTION: Total nucleic acid extraction
2. 

For HA filter extraction, place the 2-mL tubes in the bead beater.
 

SECTION: Total nucleic acid extraction
2.1. Bead beat for 150 s 
 


SECTION: Total nucleic acid extraction
2.2.  Cooldown the samples on ice for 5 min .

SECTION: Total nucleic acid extraction
2.3. Repeat Steps 9.1 and 9.2 once  .

SECTION: Total nucleic acid extraction
3. Centrifuge at maximum speed for 1 min at room temperature. 150000 rpm, 1 min 

SECTION: Total nucleic acid extraction
4. For HA filter extraction, transfer 400 µL of supernatant to the Lysis/Bind plate. 
For BCoV/BRSV/Direct extraction, transfer all supernatant to the Lysis/Bind plate.


SECTION: Total nucleic acid extraction
5. Start the protocol MN_96_Flex.bdz on the KingFisher Flex  74 min 

 


SECTION: Total nucleic acid extraction
6. Transfer the purified sample from the Elution plate to the microcentrifuge tubes.

 

SECTION: RT-ddPCR
7. Quantification by Droplet Digital PCR (ddPCR)

dx.doi.org/10.17504/protocols.io.bpg6mjze