Protocol: KingFisher SARS-CoV-2 RNA Extraction Protocol V5.

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Instrument: KingFisher Flex Purification System with 96 Deep-well Head (Cat. No. 5400630,
ThermoFisher)
Extraction Kit: Quick-DNA/RNA™ Viral MagBead (Cat. No. R2141, Zymo Research)
Effective Date: 03/14/2020
Original Author: Keith K.
Primary Reviewer: Erica C.
Last Editor: Ethan T.
Script Version: KingFisher SARS-CoV-2 RNA Extraction Script V3
Software Version: BindIt 4.0 (4.0.0.45)

Summary of SOP:

I. Equipment, Supplies, and Reagents

II. Reagent Preparation
III. Sample Preparation
IV. KingFisher Flex Reagent Plates Preparation
V. KingFisher Flex Viral DNA/RNA Extraction Procedure
VI. FAQ
VII. Index

I. Equipment, Supplies, Reagents

Equipment Catalog no. Source

KingFisher Flex with 96 Deep-Well Head 5400630 Thermo Fisher
KingFisher 96 tip comb for DW magnets 97002534 Thermo Fisher
KingFisher Deepwell 96 Plate, V-bottom, 95040450 Thermo Fisher
polypropylene
KingFisher 96 KF microplate (200μL) 97002540 Thermo Fisher

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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0
 Pipettes (P1000, P200, P10)
Filter Tips (P1000, P200, P10)
Repeat Pipette
Repeat Pipette Tips

Reagent Source Catalog no. Storage (°C)

Viral DNA/RNA Buffer Zymo Research D7020-1-200 Room Temperature
Proteinase K (lyophilized) Zymo Research D3001-2-20 Room Temperature
Proteinase K Storage Buffer Zymo Research D3001-2-20 Room Temperature
MagBinding Beads Zymo Research D4100-2-12 Room Temperature
MagBead DNA/RNA Wash 1 Zymo Research R2130-1-120 Room Temperature
(concentrate)
MagBead DNA/RNA Wash 2 Zymo Research R2130-2-80 Room Temperature
(concentrate)
DNase/RNase-Free Water Zymo Research W1001-100 Room Temperature
Ethanol 95%*** Room Temperature
Isopropanol*** Room Temperature
Beta-mercaptoethanol*** Room Temperature
*** User supplied

II. Reagent Preparation

1. Add 1 ml beta-mercaptoethanol to 200 ml Viral DNA/RNA Buffer (D7020-1-200).
2. Add 80 ml of isopropanol to the MagBead DNA/RNA Wash 1 concentrate (R2130-1-
120).
3. Add 120 ml of isopropanol to the MagBead DNA/RNA Wash 2 concentrate (R2130-2-
80).
4. Reconstitute lyophilized 20 mg Proteinase K (D3001-2-20) with 1,040 μl Proteinase K
Storage Buffer.

III. Sample Preparation

1. Swab Sample in 2 mL DNA/RNA Shield:
a) Add 10 μl of reconstituted Proteinase K to the sample
b) Vortex
c) Incubate at room temperature for 15 minutes
2. Sputum Sample in 2 mL DNA/RNA Shield:
a) Add 40 μl of reconstituted Proteinase K to the sample
b) Vortex
c) Incubate at room temperature for 15 minutes

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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0
 IV. KingFisher Flex Plates Preparation
Note: Using reagents prepared in step II
1. Sample Plate Preparation
a) Transfer 300 μl of Proteinase K treated sample in DNA/RNA Shield into a new
KingFisher Deepwell 96 Plate, V bottom (Cat. No. 95040450).
b) Add 600 μl Viral DNA/RNA Buffer to each sample.
c) Add 20 μl of MagBinding Beads to each sample.
2. Tip Plate Preparation
a) Place KingFisher 96 tip comb (Cat. No. 97002534) into a new KingFisher 96 KF
microplate (Cat. No. 97002540).
3. MagBead DNA/RNA Wash 1 Plate Preparation
a) Add 500 μl of MagBead DNA/RNA Wash 1 into a new KingFisher Deepwell 96
Plate, V bottom. Mirror the Sample Plate
4. MagBead DNA/RNA Wash 2 Plate Preparation
a) Add 500 μl of MagBead DNA/RNA Wash 2 into a new KingFisher Deepwell 96
Plate, V bottom.
5. Ethanol 1 Wash Plate Preparation
a) Add 900 μl of 95% Ethanol into a new KingFisher Deepwell 96 Plate, V bottom.
6. Ethanol 2 Wash Plate Preparation
a) Add 500 μl of 95% Ethanol into a new KingFisher Deepwell 96 Plate, V bottom.
7. Elution Plate Preparation
a) Add 50 μl of DNase/RNase Free Water into a new KingFisher 96 KF microplate.

V. Operating the KingFisher Flex:

1. Power-on the KingFisher Flex machine via the power switch on the left-hand side.
2. Log-in to connected computer and start the KingFisher software, BindIt.
3. Load the script “KingFisher SARS-CoV-2 RNA Extraction Script V3.bdz” onto the
software.
4. Click on the start button to initiate the program.
5. Follow program instructions on the KingFisher Flex Screen to load plates onto the
machine.
6. Ensure orientation of loaded plates are uniform.
7. Close KingFisher Flex plastic shield and press enter to start the script.
8. After script completion, follow instructions to remove elution plate.
9. Follow program instructions on the KingFisher Flex Screen to unload and appropriately
discard used plates.

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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0
 VI. Index
1. KingFisher Flex Plate Layout

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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0
 2. KingFisher Flex Steps

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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0
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KingFisher SARS-CoV-2 RNA Extraction Protocol V4.0