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Dilution

The Dilution protocol dilutes a sample in 5 solutions of different specified volumes.

Both the volume of the sample and the volumes of diluent are defined in the add_parameters() function to allow for user customization.

By default, 1µl of 10ng/µl sample is added to solutions containing 10, 20, 30, 40, and 50 µl of diluent, respectively.

for i in range(1, 6):
    name = "volume_" + (str) (i)
    display = "Volume " + (str) (i)
    desc = "The volume of dilutent in solution " + (str) (i) + "."
    parameters.add_float(
        variable_name=name,
        display_name=display,
        description=desc,
        default=(i*10),
        minimum=1,
        maximum=100,
        unit="µl"
    )
parameters.add_float(
    variable_name="sample_volume",
    display_name="Sample volume",
    description="The volume of the sample added to each solution.",
    default=1,
    minimum=1,
    maximum=20,
    unit="µl"
)

The first thing to do in the run() function is to define all of the labware and hardware that will be used.

An Opentrons 24 Tube Rack with Eppendorf 1.5 mL Safe-Lock Snapcap is placed in slot 3 of the deck.

tube_rack = protocol.load_labware("opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap", 3)

The diluent, water, goes into slot D2 of the tube rack.

diluent_tube = tube_rack["D2"]
diluent = protocol.define_liquid(
    name="Diluent",
    description="The liquid being used to dilute the sample.",
    display_color="#0051FF"
)
tube_rack.load_liquid(
    wells=["D2"],
    volume=150,
    liquid=diluent
)

Defining liquids isn't necessary, but it makes it easier to identify what goes where in the Opentrons app.

The sample to be diluted goes into slot D1 of the tube rack.

main_sample_tube = tube_rack["D1"]
main_sample = protocol.define_liquid(
    name="Main sample",
    description="The sample being diluted.",
    display_color="#FD9381"
)
tube_rack.load_liquid(
    wells=["D1"],
    volume=150,
    liquid=main_sample
)

A 96 20µl tip rack is placed in slot 6 of the deck.

tips = protocol.load_labware("opentrons_96_tiprack_20ul", 6)

Use the single-channel P20 pipette in the left pipette mount and specify the tip rack and starting tip.

The P20 is used instead of the P300 due to the relatively small volumes being pipetted.

left_pipette = protocol.load_instrument("p20_single_gen2", "left", tip_racks=[tips])
left_pipette.starting_tip = tips[protocol.params.starting_tip_row + protocol.params.starting_tip_col]

Now that we have all of the necessary labware and hardware, Otto can start pipetting.

The specified amounts of diluent are added to the first five slots of the tube rack.

left_pipette.pick_up_tip()
left_pipette.transfer(protocol.params.volume_1, diluent_tube, tube_rack["A1"], new_tip="never", blow_out=True, blowout_location="destination well")
left_pipette.transfer(protocol.params.volume_2, diluent_tube, tube_rack["A2"], new_tip="never", blow_out=True, blowout_location="destination well")
left_pipette.transfer(protocol.params.volume_3, diluent_tube, tube_rack["A3"], new_tip="never", blow_out=True, blowout_location="destination well")
left_pipette.transfer(protocol.params.volume_4, diluent_tube, tube_rack["A4"], new_tip="never", blow_out=True, blowout_location="destination well")
left_pipette.transfer(protocol.params.volume_5, diluent_tube, tube_rack["A5"], new_tip="never", blow_out=True, blowout_location="destination well")
left_pipette.drop_tip()

Note that we only need one tip for this process, which is why new_tip="never" is specified.

Then the specified sample volume is transferred to each of the slots of diluent.

for i in range(5):
    loc = "A" + (str) (i+1)
    left_pipette.transfer(protocol.params.sample_volume, main_sample_tube, tube_rack[loc], blow_out=True, blowout_location="destination well")