TITLE: Syngenta drug screen
AUTHORS: Ida Barlow, Adam McDermott-Rouse, Luigi Feriani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for preparing worms, preparing drug plates, dispensing worms with wormsorter and tracking on Hydra rigs</div></div>

[STEPS]
?. [Bleach worms (- 5 days)]
Bleach worms prepared for day 1 tracking:Adaptations:- remix pellet after each wash
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?. [Prepare drugs onto plates (-1 days)]
Label each DESTINATION plate with the source plate the DESTINATION SLOT in the opentrons robot.
?. [Prepare drugs onto plates (-1 days)]
Prepare drug source plates in skirted 96 well plate so that each column corresponds to a single drug at a single concentration. The final concentration of DMSO should be 0.1%.
?. [Prepare drugs onto plates (-1 days)]
Run opentrons robot using prepared protocol.
NB.- One drug source plate with each column corresponding to a drug for use with a multichannel pipette.- Destination and source plate locations are specified.- The opentrons robot will randomise the column locations from each source plate to a destination plate- The date is used as the random seed
?. [Prepare drugs onto plates (-1 days)]
Protocol parameters:# multichannel pipette parameters and tipracksmulti_pipette_type = 'p10-Multi'multi_pipette_mount = 'left'tiprackdrugs_slots = ['3']tiprackdrugs_type = 'opentrons-tiprack-10ul'tiprackdrugs_startfrom = '1'tiprackH2O_slot = '6'tiprackH2O_type = 'opentrons-tiprack-10ul'# tiprackH2O_startfrom = '1'# water troughH2O_source_slot = '9'H2O_source_type = 'trough-12row'H2O_source_well = 'A1'H2O_volume = 5# drugs sourcedrugs_source_slots = '2'#,'5','8','11']drugs_source_type = '96-well-plate-pcr-thermofisher'frombottom_off = +0.3 # mm from bottom of src wellsdrugs_volume = 3# destination platesagar_thickness = +3.7 # mm from the bottom of the well for 200ul agar per welldestination_slots = ['1','4','7', '5']destination_type = '96-well-plate-sqfb-whatman'n_columns = 12# create mapping from sources to destination.seed = 20191031 # for reproducibility. Let's use the experimental date for the actual experiment, something else for debuggingnp.random.seed(seed)
?. [Prepare drugs onto plates (-1 days)]
Make sure all labware is loaded correctly
?. [Prepare drugs onto plates (-1 days)]
Run protocol and monitor robot to ensure all the tips are removed
?. [Prepare drugs onto plates (-1 days)]
Tip racks have to replaced after each destination plate is filled
?. [Prepare drugs onto plates (-1 days)]
Allow the plates to dry for 30 minutes
?. [Seed plates (-1 days)]
Use VIAFILL dispenser to dispense  1:10 diluted OP50 into each well of each drug plate
5 µl
?. [Seed plates (-1 days)]
Allow to dry for 30 minutes under the flow hood
?. [Seed plates (-1 days)]
Keep at room temperature over night (covered in the dark as some drugs are light-sensitive)
?. [Disepensing worms]
Wash worms off the 150mm plates with M9 using a pasteur pipette into 15ml falcons
?. [Disepensing worms]
Spin at  for 2 minutes to pellet the worms
Centrifuge: 1500 33
?. [Disepensing worms]
Remove supernatent and fill M9
?. [Disepensing worms]
Repeats steps 12-13 two more times
?. [Disepensing worms]
After final wash fill falcon with M9, transfer contents of 15mL falcons to 50mL and fill up to 45mL with M9.
?. [Disepensing worms]
Use wormsorter to dispense 2 worms per well
?. [Disepensing worms]
Allow liquid to dry off the plates for 30 mins under the flow hood
?. [Disepensing worms]
Incubate in drug for 4 hours
?. [Disepensing worms]
Hydra tracking : 15 mins 25fps, exposure 25000msec
?. [Pick L4 worms for bleaching (-9 days)]
Pick L4 worms onto 4 x  OP50-seeded NGM 90mm plates (10 worms per plate) for day 1 of tracking
?. [Refeed L1s (- 3 days)]
At 17:00 Refeed L1s for day 1 tracking onto 5 x 150mm plates as in protocol above by dropping 4 small droplets around the edges of the plate (off food). Wash worms once before refeedingKeep worms at
20 °C
?. [Refeed L1s (- 2 days)]
At 17:00Refeed L1s for day 2 tracking onto 5 x 150mm plates as in protocol above by dropping 4 small droplets around the edges of the plate (off food). Wash worms once before refeedingKeep worms at
20 °C
?. [Bleach worms (- 4 days)]
Bleach worms prepared for day 2 tracking and keep at   in rotatotor until refeeding:Adaptations:- remix pellet after each wash
20 °C
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?. [Pick L4 worms for bleaching and pour 96WPs (- 8 days)]
Once agar has cooled and salts have been added pour agar into 65 x 96 well plates using VIAFILL dispenser. Dispense   per well. Once cooled, store lidside down in airtight container at  .
200 µl
4 °C
?. [Pick L4 worms for bleaching and pour 96WPs (- 8 days)]
Prepare 1.5L low peptone NGM and autoclave
?. [Pick L4 worms for bleaching and pour 96WPs (- 8 days)]
Pick L4 worms onto 4 x  OP50-seeded NGM 90mm plates (10 worms per plate) for day 2 of tracking
?. [Dry 96 well plates (-2 days)]
Weigh 3 random plates
?. [Dry 96 well plates (-2 days)]
Place in flow hood for 3 hours to allow to dry (with lids off)
?. [Dry 96 well plates (-2 days)]
Weigh 3 random plates
?. [Prepare drugs onto plates (-1 days)]
Use VIAFLO to fill 30 x SOURCE plates with  water and drug from STOCK plates.
7 µl
0.5 µl