Nanotrap® KingFisher™ Concentration/Extraction & MagMAX KingFisher™ Extraction
Christine Moe, Jamie VanTassell, Julia Raymond, Marlene K Wolfe, Orlando Sablon III, Pengbo Liu
Nanotrap
KingFisher
Ceres
nano
magnetic virus particles
magnetic
automated
wastewater
SARS-CoV-2
COVID-19
Abstract
These protocols have been adapted from Ceres "Nanotrap® Wastewater Protocol using MagMAX Kits" (APP-030, Revision 0, Nov 2021).
This protocol uses Nanotrap® Magnetic Virus Particles and Nanotrap® Enhancement Reagent 1 (ER1) to capture and concentrate viruses in wastewater samples. It is optimized for viral capture from 10 mL samples of wastewater and is compatible with three nucleic acid extraction kits from ThermoFisher. This protocol has been used and adapted for SARS-CoV-2 viral capture at Emory University for wastewater surveillance of COVID-19.
Steps
Ceres Nanosciences Nanotrap® KingFisher™ Procedure
Label two KingFisher™ 24 Well Deep Well sample plates “Sample Plate 1” and “Sample Plate 2”. You will split the total sample volume to process across the two plates for each sample.
Equipment
| Value | Label |
|---|---|
| Plastics for 24 deep-well format | NAME |
| KingFisher | BRAND |
| 95040470 | SKU |
Aliquot 10µL of Bovine Respiratory Syncytial Virus ( BRSV )
Aliquot 4865µL of wastewater sample to one well (one well per sample) of Sample Plate 1 (the new KingFisher™ 24 Well Deep Well sample plate).
Aliquot another 4865µL of wastewater sample to the same well on Sample Plate 2 (a second KingFisher™ 24 Well Deep Well sample plate).
For example, if you loaded a sample into well A1 of Sample Plate 1, load the second volume of that sample into well A1 of Sample Plate 2.
Designate the same well on Sample Plate 1 and Sample Plate 2 as a negative control. Add 4865µL of
Aliquot 50µL of
Incubate the sample plates for 0h 10m 0s at 20°C
Aliquot 75µL of
Insert the KingFisher™ 24 well-KF Deep Well Comb into Sample Plate 1.
Equipment
| Value | Label |
|---|---|
| Plastics for 24 deep-well tip comb and plate format | NAME |
| KingFisher | BRAND |
| 97002610 | SKU |
Prepare the Lysis Plate by aliquoting 500µL of
Run NT KingFisher™ Script
Once the protocol is completed, the KingFisher™ plate to which lysis solution was added will contain 500µL of lysate that is ready to be run on the MagMAX KF extraction procedure.
MagMAX KingFisher™ Extraction Procedure
Label four KingFisher™ 96 Deep Well Plate-Sample Plate wells "Wash 1", "Binding", "Wash 2", and "Elution".
Prepare Wash 1 Plate by aliquoting 1mL of MagMAX Wash Buffer to a new KingFisher™ 96 Deep Well Plate matching the number and location of the KingFisher™ 96 Deep Well Plate-Sample Plate wells.
Prepare the Elution Plate by aliquoting 60µL of MagMAX Elution buffer to a new KingFisher™ 96 - 200 µL plate matching the number and location of the “Sample Plate” wells.
To prepare the Binding Plate , obtain a new KingFisher™ 96 Deep Well Plate.
Aliquot 10µL of MagMAX Proteinase K to each well in the Binding Plate in which lysate will be added.
Aliquot 400µL of the lysate from each well of the lysis plate used in Part 1 of the protocol into the Binding Plate.
Aliquot 530µL of MagMAX Binding Solution into each deep well which lysate was added in the Binding Plate.
Vortex and thoroughly mix the MagMAX DNA/RNA Binding Beads.
Aliquot 20µL of vortexed binding beads to each well in which lysate was added in the Binding Plate.
Insert the KingFisher™ 96 Deep Well Comb into the Binding Plate.
Prepare Wash Plate 2 by aliquoting 1mL of 80% EtOH to a new KingFisher™ 96 Deep Well Plate matching the number and location of the KingFisher™ 96 Deep Well Plate-Sample Plate wells.
Run MagMAX KingFisher™ Protocol
Once the protocol is completed, the KingFisher™ 96-Elution Plate will contain ~60 µL purified viral RNA that is ready to be loaded onto a PCR plate or 1.7 mL RNA tube.
