SARS-CoV-2 RNA extraction with Ceres Nanotrap and Zymo Environ Water
Padmini Ramachandran, Tamara Walsky, Amanda Windsor, Maria Hoffmann, Christopher Grim, Kathryn Judy
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Abstract
This protocol uses the Ceres Nanotrap® particle-based virus capture and concentration method for 10mL of wastewater followed by extraction with the Zymo Environ™ Water RNA extraction kit with a Zymo DNase step.
Before start
Store the reagents separately from RNA/TNA (total nucleic acid) samples. * Use a clean designated work area and separate pipettes for pre- and post-extraction steps to minimize the potential for cross-contamination
- Wear a lab coat and protective eyewear.
- Wear gloves and change them often.
- Prevent contamination by using aerosol-resistant pipette tips.
Steps
Before you start
Turn on heat block`95°C`
Ensure appropriate volume of DNase I is available (5µL per sample), or make new aliquots
Add 275µL DNase/RNase-Free water to reconstitute lyophilized DNase I (1U/µL)
Viral Capture with Nanotrap® Particles
Shake wastewater bottle to mix then let sit 0h 0m 45s
Using a 10mL serological pipette, carefully pipette 10mL of wastewater into a 15mL conical tube
Add 100µL of Nanotrap® Enhancement Reagent 2 (ER2) and invert 15mL tube 2-3 times to mix
Re-suspend Nanotrap® particles by inverting the bottle 5 times
Add 150µL Nanotrap® particles to the sample
Incubate samples Room temperature 0h 10m 0s with constant rotation
Place samples on magnetic rack to separate Magnetic Nanotrap® particles from the sample - at least 0h 2m 0s
After beads have settled, use a 5mL serological pipette to remove all of the supernatant without disturbing the pelleted beads
Add 1mL of DNAse/RNAse Free water to the tube
Remove tube from magnet and re-suspend the pelleted beads using a 100-1000uL pipette
Transfer suspended beads to a 1.5mL microcentrifuge tube
Place microcentrifuge tube on the 2mL tube-compatible magnetic rack
Incubate until the beads have settled - at least 0h 2m 0s
Remove supernatant with a 100-1000uL pipette without disturbing the pellet. Remove any small amount of remaining supernatant with a smaller pipette tip (e.g. 2-20uL pipette)
Remove the tubes from the magnet and re-suspend the pellet with 375µL Zymo DNA/RNA Shield and 125µL Zymo DNase/RNase-Free water from the Zymo Environ Water RNA Kit
Incubate the samples at 95°C for 0h 5m 0s
While samples are incubating, add 400µL Zymo RNA Binding Buffer from the Zymo Environ Water RNA Kit to new 1.5mL tubes (one per sample)
Remove tubes from heat block and place on a magnetic rack and allow beads to settle until supernatant is clear - at least 0h 2m 0s
Reset heat block temperature to 27°C
Transfer 400µL of supernatant to the corresponding tube prepared in step 15.1 and mix by gentle pipetting
Zymo Environ™ Water RNA Kit
Transfer entire sample to a Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge 10000x g and keep the flow-through
Add 800uL of ethanol (95-100%) to the flow-through in the collection tube from step 18 and mix well by gentle pipetting
Transfer 800µL into a new Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge 10000x g and discard the flow through
Repeat step 19 with the remaining 800µL of sample using the same collection tube
Add 400µL of RNA Prep Buffer to the column and centrifuge 10000x g and discard the flow-through
Transfer column to an RNase-Free 1.5mL microcentrifuge tube
Add 100µL Zymo DNase/RNase-Free water directly to the column matrix and centrifuge 10000x g and keep the flow-through for step 24
Place a Zymo-Spin III-HRC Filter into a new collection tube and add 600µL Prep Solution Centrifuge 8000x g discard the flow-through
transfer the column to an RNase-Free 1.5mL microcentrifuge tube
Transfer the eluted RNA from step 22 into the Zymo-Spin III-HRC filter prepared in step 23 and 16000x g keep the flow-through
Add 200µL RNA Binding Buffer to the filtrate and mix well by gently pipetting up and down.
Add 300µL of ethanol (95-100%) to the filtrate + RNA Binding Buffer and mix well by gently pipetting up and down.
Transfer the mixture into a Zymo-Spin IC column in a collection tube and 10000x g discard the flow-through
Add 400µL RNA Wash Buffer to column and 10000x g Discard flow-through
Add 5µL DNase I and 35µL DNA Digestion Buffer to the column matrix
Incubate 27°C 0h 20m 0s
Add 400µL of RNA Prep Buffer to the column and 10000x g discard the flow-through.
Add 700µL of RNA Wash Buffer to the column and 10000x g discard the flow-through.
Add 400µL of RNA Wash Buffer to the column and 10000x g to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
Add 50µL of DNase/RNase-Free Water directly to the column matrix
and 10000x g The eluted RNA can be used immediately or stored at -70°C.
