TITLE: Opentrons COVID-19 testing (Zymo, station  B, 24 samples)
AUTHORS: Max Marrone

[DESCRIPTION]
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[STEPS]
?. [Station B: Initial OT-2 setup]
Clean the OT-2.
?. [Station B: Initial OT-2 setup]
Place the following labware on the OT-2's deck:Slot 1: An Opentrons Temperature Module with an Opentrons 96 well aluminum block and an empty, sterile NEST 100 µL PCR plate.Slots 3 and 6: A full, sterile rack of Opentrons 200 µL filter tips.Slot 4: An Opentrons Magnetic Module with nothing on it.Slot 11: An empty NEST 1-well reservoir, for organic liquid waste.
?. [Station B: Initial OT-2 setup]
Start pre-cooling the Temperature Module to .
6 °C
?. [Station B: Sample preparation]
Prepare the sample well plate.The sample well plate is the NEST 96 deep well plate that was output from Station A.The 24 samples (#1–#24) should be laid out like this:  ABCDEFGHIJKLM1Col. 1Col. 2Col. 3Col. 4Col. 5Col. 6Col. 7Col. 8Col. 9Col. 10Col. 11Col. 122Row A#1empty#9empty#17emptyemptyemptyemptyemptyemptyempty3Row B#2empty#10empty#18emptyemptyemptyemptyemptyemptyempty4Row C#3empty#11empty#19emptyemptyemptyemptyemptyemptyempty5Row D#4empty#12empty#20emptyemptyemptyemptyemptyemptyempty6Row E#5empty#13empty#21emptyemptyemptyemptyemptyemptyempty7Row F#6empty#14empty#22emptyemptyemptyemptyemptyemptyempty8Row G#7empty#15empty#23emptyemptyemptyemptyemptyemptyempty9Row H#8empty#16empty#24emptyemptyemptyemptyemptyemptyemptyEach sample well should already contain a sample, Proteinase K, and internal extraction control RNA, mixed.
ABCDEFGHIJKLM1Col. 1Col. 2Col. 3Col. 4Col. 5Col. 6Col. 7Col. 8Col. 9Col. 10Col. 11Col. 122Row A#1empty#9empty#17emptyemptyemptyemptyemptyemptyempty3Row B#2empty#10empty#18emptyemptyemptyemptyemptyemptyempty4Row C#3empty#11empty#19emptyemptyemptyemptyemptyemptyempty5Row D#4empty#12empty#20emptyemptyemptyemptyemptyemptyempty6Row E#5empty#13empty#21emptyemptyemptyemptyemptyemptyempty7Row F#6empty#14empty#22emptyemptyemptyemptyemptyemptyempty8Row G#7empty#15empty#23emptyemptyemptyemptyemptyemptyempty9Row H#8empty#16empty#24emptyemptyemptyemptyemptyemptyempty
?. [Station B: Reagent preparation]
Prepare 3x ...
[Viral DNA/RNA Buffer + MagBinding Beads]
?. [Station B: Reagent preparation]
In a sterile 15 mL tube, add .
[Viral DNA/RNA Buffer]
?. [Station B: Reagent preparation]
Vortex the MagBinding Beads in their container.
?. [Station B: Reagent preparation]
Pipette  to the 15 mL tube.
[vortexed MagBinding Beads]
?. [Station B: Reagent preparation]
Repeat for a total of 3x .
[Viral DNA/RNA Buffer + MagBinding Beads]
?. [Station B: Reagent preparation]
Wait for the Temperature Module to reach .
6 °C
?. [Station B: Reagent preparation]
Prepare the reagent reservoir.Into a 15 mL NEST 12 well reservoir, pipette or pour the following reagents:Well 1:   (2 prepared tubes' worth)Well 2:   (1 prepared tube's worth)Well 3: EmptyWell 4:  Well 5: EmptyWell 6:   Well 7: EmptyWell 8:   Well 9:   Well 10: EmptyWell 11: EmptyWell 4: Well 5: EmptyWell 6:   Well 7: EmptyWell 8:   Well 9:   Well 10: EmptyWell 11: EmptyWell 6: Well 7: EmptyWell 8:   Well 9:   Well 10: EmptyWell 11: EmptyWell 8: Well 9: Well 10: EmptyWell 11: EmptyWell 12:
This step is time-sensitive. The MagBinding Beads will slowly settle in the Viral DNA/RNA Buffer. Continue quickly until you start the run on the OT-2. Vortex the Viral DNA/RNA Buffer + MagBinding Beads immediately before adding them to the reservoir.
[Viral DNA/RNA Buffer + MagBinding Beads]
[Viral DNA/RNA Buffer + MagBinding Beads]
[MagBead DNA/RNA Wash 1]
[MagBead DNA/RNA Wash 2]
[95-100% ethanol]
[95-100% ethanol]
[nuclease-free water]
?. [Station B: Final OT-2 setup]
Place the sample well plate in slot 4.
?. [Station B: Final OT-2 setup]
Place the reagent reservoir in slot 2.
?. [Station B: Final OT-2 setup]
Double-check all the labware to make sure it looks correct.Check that labware are inserted the right way around (well A1 at the top-left for the plates, well 1 at the left for the troughs).Check that labware are properly clicked into the deck slots.Check the deck layout.
?. [Station B: Running the robot]
Run the Station B protocol on the OT-2. The protocol file should be StationB-24samples-Zymo.py.
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?. [Station B: Running the robot]
Wait for the run to finish.
?. [Station B: Running the robot]
The output is the NEST 100 µL PCR plate sitting on the aluminum block atop the Temperature Module in slot 1.