SW-2 SWAB PROCESSING

Add 500µL of ATL-DX buffer and 20µL Proteinase K to the bead tubes prepared in Step 4 of Before Start section under the Guidelines & Warning tab.

Place the sample swab in the bead tube with ATL buffer and Proteinase K. Use sterile scissors to carefully cut off the swab heads or transfer the pre-cut swab head from the original container to the bead tube with forceps.

Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material, 100µL EBV, and 100µL ZIKV standard into a tube from Step 3 of Before Start section. Add 250µL ATL-DX buffer and 20µL proteinase K.

Include a negative control for each batch of samples: a bead tube from Step 3 of Before Start section with 500µL ATL-DX buffer and 20µL proteinase K.

Incubate samples in a thermomixer at 1400rpm.

Refill the dry ice compartment of the Bullet Blender if necessary. After incubation, load all samples into the Bullet Blender.

Set the speed at 12 and the time at 3. Press Start.

Let the samples settle for 0h 1m 0s in the Bullet Blender and then repeat Step 7.

Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.

Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.

Set up the Tip Comb, Wash, and Elution Plates outside the instrument according to the following table.

A	B	C	D	E
Plate ID	Plate position	Plate type	Reagent	Volume per well
Tip comb	7	Place a 96 Deep-well Tip comb in a deep-well plate
Elution	6	Deep-Well	Nuclease-free water	75 µL
Wash 4	5	Deep-Well	100% ethanol	750 µL
Wash 3	4	Deep-Well	80% ethanol	750 µL
Wash 2	3	Deep-Well	Buffer AW2	700 µL
Wash 1	2	Deep-Well	Buffer AW1	700 µL
Sample	1	Sample Lysate	Lysate and lysis buffer	985 µL

Centrifuge the bead tubes with lysate from Step 8 for 12000x g.

Transfer 290µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.

Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix; avoid foaming (it can be mixed on a Hula mixer for 2 min). Add 695µL mixture to each sample. Each well including sample lysate should be 985µL.

Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.

Start the run, then load the prepared plates into position when prompted by the instrument.

After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

In a 0.6mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions (Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit) (see Appendix 2 and Appendix 3).

Proceed with sample testing following the REDI-NET SOP SW-4 Swab Testing or store at -20°C for less than 2 weeks (for long-term storage the sample needs to be stored at -80°C following the REDI-NET SOP SW-3 Swab Storage).

Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.

Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.

Set up the Sample Plate according to the table below:

A	B	C	D
Row ID	Plate Row	Reagent	Volume per well
Sample row	A	Lysate and lysis buffer	985 µL
Wash 1	B	Buffer AW1	700 µL
Wash 2	C	Buffer AW2	700 µL
Wash 3	D	80% ethanol	750 µL
Wash 4	E	100% ethanol	750 µL
Tip Comb	F	Tip comb
****	G	Empty
****	H

Set up the Elution Strip according to the table below:

A	B	C	D
Strip ID	Row	Reagent	Volume per well
Elution	A	Nuclease-free water	75 µL

Centrifuge the bead tubes with lysate from Step 8 for 12000x g.

Transfer 290µL supernatant without any particle carryover to the wells of the Deep-well Row A. This row becomes the Sample Row.

Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the Sample Plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix and avoid foaming. Add 695µL mixture to each sample. Each well including sample lysate should be 985µL.

Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.

Start the run, then load the prepared plate and Elution Strip into position when prompted by the instrument.

After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

In a 0.6mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions (Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit) (see Appendix 2 and Appendix 3).

Proceed with sample testing following the REDI-NET SOP SW-4 Swab Testing or store at -20°C for less than 2 weeks (for long-term storage the sample needs to be stored at -80°C following the REDI-NET SOP SW-3 Swab Storage).

DNA quantification:

According to the volume of sample used, add the 1x HS dsDNA Qubit Assay for a final volume of 200µL (i.e., if using 3µL of sample, add 197µL of 1x HS dsDNA Qubit Assay). Vortex for 5 - 10 seconds, then incubate for 0h 2m 0s at Room temperature

RNA Quantification:

In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:

A	B	C
Reagents	Volume/sample	Volume for n+1 sample
Qubit RNA HS Assay buffer	199 µL	…. µL
Qubit RNA HS Assay Dye	1 µL	…. µL

In a new 0.6ml tube, mix 197µL of Qubit HS RNA Assay working solution and 3µL of the sample. Vortex for 5 - 10 seconds, then incubate for 0h 2m 0s at Room temperature before reading.