TITLE: Ceres Nanoparticles Concentration and Extraction using MagMAX Wastewater kit
AUTHORS: Shannon Fitz, Alex Shaw, Dilip Abraham, michael Owusu

[DESCRIPTION]
Ceres Nanotrap Microbiome A Particles are used to capture and concentrate important pathogens from samples. https://www.ceresnano.com/protocols Protocol APP-082 was used to concentrate and extract target pathogens, scaled up from 10 mL to 35 mL samples using Ceres Microbiome A particles and the MagMAX Wastewater Ultra Nucleic Acid Isolation Kit. Following discussions with Ceres scientists, Enhancement Reagent 3 is recommended as opposed to Enhancement Reagent 1. 

[STEPS]
SECTION: Concentration and extraction
1. Manual Ceres Nanotrap concentration using Nanotrap Microbiome A Particles for a 35 mL environmental sample
SECTION: Concentration and extraction
1.1. Invert the environmental water sample 5 times to mix. Then, let it sit for 45 seconds at room temperature. (No need to wait for samples to reach room temperature before processing). 
SECTION: Concentration and extraction
1.2. Agitate, then filter environmental water sample through a coffee filter to remove suspended solids. Transfer 35 mL of filtered sample into a clean 50 mL conical tube.
SECTION: Concentration and extraction
1.3. Add 100 µL of Nanotrap Enhancement Reagent 3 (ER3) to the sample, cap the tube, and then invert 2 times to mix.
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1.4. Add 525 µL of Nanotrap Microbiome A Particles (Nanotrap particles) to the sample, cap the sample and then invert 2 times to mix the particles.
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1.5. Incubate samples with Nanotrap particles at room temperature for 30 minutes.

Note: Invert every 5 minutes or use a rotator.
SECTION: Concentration and extraction
1.6. Place the tube on a DynaMag-50 magnetic rack to separate the Nanotrap particles from the sample for 10 minutes.
SECTION: Concentration and extraction
1.7. Using a serological pipette, discard the supernatant carefully without disturbing the Nanotrap particle pellet.

Note: Can use a P-1000 or P-200 pipette to remove any remaining supernatant from the sample (be careful to not lose any Nanotrap particles when removing supernatant).
SECTION: Concentration and extraction
1.8. Add 1 mL of Nanotrap Buffer 2 to the tube and re-suspend the Nanotrap particle pellet by pipetting on the walls of the conical tube, gently re-suspend until all Nanotrap particles have been completely collected.
SECTION: Concentration and extraction
1.9. Transfer the Nanotrap particle suspension to a new 2 mL microcentrifuge tube.
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1.10. Place the 2 mL microcentrifuge tube on a DynaMag-2 magnetic rack to separate the Nanotrap particles from the sample for 2 minutes.
SECTION: Concentration and extraction
1.11. Using a P-1000 pipette, discard the supernatant carefully without disturbing the Nanotrap particle pellet.

Note: If any small amount of liquid is still present, use a smaller pipette to remove all the supernatant from the bottom of the tube. 
SECTION: Concentration and extraction
1.12. Add 500 µL of MagMAX‱ Microbiome Lysis Solution to Nanotrap particle pellet, pipette up and down until Nanotrap particles are resuspended completely.
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1.13. Close the tube lid and incubate the samples on a heating block at 56°C for 10 minutes.
SECTION: Concentration and extraction
1.14. Place the 2 mL microcentrifuge tube on a DynaMag-2 magnetic rack to separate the Nanotrap particles from the sample for 2 minutes.

Note: May need to briefly centrifuge the tube (Mini Centrifuge at 2000 g for 2-5 seconds) to remove drops from inside the lid before magnetic separation.
SECTION: Concentration and extraction
1.15. Transfer 400 µL of supernatant/lysate to a new 2 mL collection tube and discard the Nanotrap particles pellet.
SECTION: Concentration and extraction
1.16. Sample is now ready for Part 2.
SECTION: Concentration and extraction
1.17. Part 2: Nanotrap Microbiome A MagMAX Wastewater Extraction Procedure
SECTION: Concentration and extraction
1.18. Add 530 µL of MagMAX Binding Buffer to the sample/lysate.
SECTION: Concentration and extraction
1.19. Add 10 µL of MagMAX Proteinase K to the sample/lysate.
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1.20. Add 20 µL of MagMAX Magnetic Beads to the sample/lysate.
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1.21. Vortex to mix, then incubate at 65⁰C on a heat block for 10 minutes.
SECTION: Concentration and extraction
1.22. Place the tube on a DynaMag-2 magnetic rack to separate the magnetic beads from the sample for 2 minutes, then discard the supernatant using a pipette.

Note: May need to briefly centrifuge the tube (Mini Centrifuge at 2000 g for 2-5 seconds) to remove drops from inside the lid before magnetic separation.
SECTION: Concentration and extraction
1.23. Add 1000 µL of MagMAX wash buffer to sample and re-suspend the magnetic beads using a pipette.
SECTION: Concentration and extraction
1.24. Place the tube on a DynaMag-2 magnetic rack to separate the magnetic beads from the sample for 2 minutes, then discard the supernatant.
SECTION: Concentration and extraction
1.25. Add 1000 µL of 80% EtOH to sample and re-suspend the magnetic beads using a pipette. 
SECTION: Concentration and extraction
1.26. Place the tube on a DynaMag-2 magnetic rack to separate the magnetic beads from the sample for 2 minutes, then discard the supernatant.
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1.27. Add 500 µL of 80% EtOH to sample and re-suspend the magnetic beads using a pipette.
SECTION: Concentration and extraction
1.28. Place the tube on a DynaMag-2 magnetic rack to separate the magnetic beads from the sample for 2 minutes, then discard the supernatant by using a pipette.
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1.29. Centrifuge the tube (Mini Centrifuge at 2000 g for 30 seconds).
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1.30. Place the tube on a DynaMag-2 magnetic rack, then remove excess 80% EtOH using a smaller pipette.
SECTION: Concentration and extraction
1.31. Add 100 µL of MagMAX Elution Solution to re-suspend the magnetic beads and then incubate at 65⁰C for 10 minutes on a heat block (close caps).
SECTION: Concentration and extraction
1.32. Place the tube in the DynaMag-2 magnetic rack to separate the magnetic beads from the sample for 2 minutes.

Note: May need to briefly centrifuge the tube (Mini Centrifuge at 2000 g for 2-5 seconds) to remove drops/condensation from inside the lid before magnetic separation.
SECTION: Concentration and extraction
1.33. Transfer the supernatant to a new tube, the sample is ready for downstream analysis or can be stored at -80⁰C.

Note: Multiple freeze-thaw cycles may cause degradation.