Manual Nanotrap Concentration and RNA Extraction for SARS-CoV-2 Viral Capture

Place a 50 mL conical tube in a tube rack.

Vigorously shake the sample to re-suspend solids. Immediately move on to Step 3.

Add 50mL of wastewater to the conical tube inside the biosafety cabinet.

Transfer 40mL of the top supernatant into a new conical tube.

Add 10µL of BRSV to the conical tube.

Add 400µL of to the sample and invert 2-3 times to mix together and to create a 10:1 sample volume to particle ratio.

Invert samples 3-4 times every 5 minutes at Room temperature for 0h 20m 0s.

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Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow the sample to sit in the rack for at least 0h 10m 0s.

Use a pipette to discard the supernatant. Be careful not to disturb the red pellet of Nanotrap particles.

Add 1mL of to the conical tube. Vortex the tube to re-suspend the pellet.

Transfer liquid to a 1.7mL tube. Place tubes on magnetic rack and allow to sit for 0h 2m 0s.

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Remove and discard the supernatant. Do not disturb the pellet.

Add 140µL of 1X PBS to the particle pellet.

In a separate 1.7 mL tube, add 560µL of and 5.6µL of . Mix well by pipetting up and down several times.

In a regular, non-magnetic rack, add the Buffer AVL and carrier RNA to the particle pellet. Suspend the pellet by using the pipette to wash the sides of the conical tube with the lysis buffer, carrier RNA, and 1X PBS mixture.

Incubate the sample at Room temperature for 0h 10m 0s.

Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow sample to sit on the rack for 0h 2m 0s.

Transfer the supernatant to a 1.7 mL tube and discard the pellet.

Add 560µL of 96% to sample. Mix well by pipetting up and down several times.

Add 630µL of the sample mix onto a QIAamp Mini column.

Centrifuge sample at full speed for 0h 1m 0s. Once complete, discard the filtrate.

Repeat Step 26 until all of the sample has gone through the column.

Transfer the QIAamp Mini Column into a new collection tube, and discard the tube containing the filtrate.

Open the QIAamp Mini Column and add 500µL of . Centrifuge the sample at full speed for 0h 1m 0s. Once complete, discard the filtrate.

Open the QIAamp Mini Column and add 500µL of . Centrifuge the sample at full speed for 0h 3m 0s. Once complete, discard the filtrate.

Place the QIAamp Mini Column into a new collection tube and discard the old one containing filtrate. Centrifuge the sample at full speed for 0h 1m 0s to remove any buffer AW2 carryover.

Place the QIAamp Mini Column into a final, labeled tube and discard the old one containing the filtrate

Open the QIAamp Mini Column and add 60µL of and incubate at Room temperature for 0h 3m 0s.

Centrifuge the sample at 16000rpm,0h 0m 0s for 0h 2m 0s.

Separate sample into at least two aliquots. Store in -80°C freezer until it is ready for PCR.

Begin making the 10X PBS Solution by mixing the following:

• 80g NaCl
• 2g KCl
• 14.4g Na₂HPO₄
• 2.4g KH₂PO₄
• 800mL Ddwater

Adjust the solution to 7.4 by adding 5% NaOH and/or 5% HCl.

Add more until the volume is 1000mL.

Dilute the PBS solution by adding 100mL of 10X PBS to 900mL distilled water to create 1X PBS.

Dispense mixture into aliquots and autoclave for 0h 20m 0s. Store at room temperature.