Liquid Handling Guide

Choose commands from the physical behavior the assay needs, not from which call is shortest to write. Every command still depends on correct liquid volumes, labware geometry, pipette range, tips, and contamination controls.

Command Layers

Building-block commands

Use when aspiration and dispensing need independent control:

pipette.pick_up_tip()
pipette.aspirate(50, source.bottom(z=1), flow_rate=25)
protocol.delay(seconds=1)
pipette.dispense(50, destination.bottom(z=2), flow_rate=20, push_out=5)
pipette.blow_out(destination.top(z=-1))
pipette.drop_tip()

Advantages:

• Explicit position and order.
• Independent flow rates and delays.
• Fine control for viscous, volatile, foaming, low-volume, or bead workflows.

Costs:

• The author owns tip state and volume state.
• More opportunities to aspirate with no tip, overfill the pipette, or leave residual volume.

Standard complex commands

Use for conventional source-to-destination mappings:

pipette.transfer(
    volume=50,
    source=source_plate.wells()[:8],
    dest=destination_plate.wells()[:8],
    new_tip="always",
    mix_after=(3, 30),
)

• transfer(): one or more source-to-destination transfers.
• distribute(): one source to many destinations, normally with an excess disposal volume.
• consolidate(): many sources into one destination.

Inspect the simulation run log. Complex commands expand into many building blocks, and their expansion changes with parameters and API level.

Liquid-class complex commands, Flex API 2.24+

Use an Opentrons-verified class when the pipette/tip combination is supported and the liquid resembles the verified model:

viscous = protocol.get_liquid_class("glycerol_50")

pipette.transfer_with_liquid_class(
    liquid_class=viscous,
    volume=50,
    source=reservoir["A1"],
    dest=plate["A1"],
    new_tip="always",
    trash_location=trash,
)

Related methods:

• distribute_with_liquid_class()
• consolidate_with_liquid_class()

Verified classes include water, 80% ethanol, and 50% glycerol. A liquid class controls multiple coupled properties such as flow rate, submerge and retract behavior, delays, air gaps, positions, and push-out. Do not casually override one property without testing the full result.

Opentrons-verified liquid classes are for supported Flex pipette and tip combinations, not OT-2 pipettes.

Source and Destination Mapping

Complex commands accept a single well or a sequence. Make the intended mapping explicit:

• One source, one destination: one transfer.
• One source, many destinations: repeated transfers or a distribution.
• Many sources, one destination: repeated transfers or a consolidation.
• Equal-length source and destination lists: pairwise transfers.

Do not assume row-major ordering:

# Explicit sample order is easier to audit.
sample_wells = [plate[name] for name in ("A1", "B1", "C1", "D1")]

For multi-channel pipettes, the referenced well anchors the pipette's primary channel:

• A full 8-channel pipette normally targets an entire column by referencing its A-row well.
• A full 96-channel pipette addresses an entire 96-well rack or plate.
• A partial-column layout has a different primary channel; follow the layout documentation rather than reusing full-column assumptions.

Tip Policy

new_tip is a contamination decision.

For standard distribute() and consolidate(), new_tip="always" still uses one tip for the entire command, including refills. The isolation behavior below applies to transfer(); use explicit transfers or building blocks for independent samples. Liquid-class commands have their own policies.

For a shared reagent:

• Aspirating repeatedly from the same source with one tip may be acceptable only if the tip never contacts incompatible destination liquid.
• A submerged dispense can wet the exterior or interior of the tip.
• Touch tip, mix, or bottom-contact dispense increases contamination risk.
• Controls and samples generally require separate tips.

Calculate tips for every conditional path. Multi-channel operations consume sets, not individual command calls.

Flow Rate, Position, and Delays

Flex 50 µL volume modes

The nominal 1–50 µL range spans two modes. With an empty Flex 1- or 8-channel 50 µL pipette, call configure_for_volume() before pickup:

pipette.configure_for_volume(1)
pipette.pick_up_tip()
pipette.aspirate(1, source)
pipette.dispense(1, destination)
pipette.drop_tip()

Inputs 1–4.9 µL select a 1–30 µL mode; inputs 5–50 µL select a 5–50 µL mode. Tips may further limit capacity. Reconfigure while empty when changing modes, and requalify low-volume delivery for the actual liquid. Simulation validates the mode/range contract, not accuracy at 1 µL.

The 10.0.0 simulator reports min_volume=0.5 in low-volume mode, below the official guide's 1 µL lower limit. Keep the documented 1 µL limit unless Opentrons explicitly qualifies a lower range for the exact hardware and tips; the property alone is not evidence of accuracy below 1 µL. Mode changes also reset flow rates, so apply any validated custom rate after changing mode.

Relative and absolute rates

rate= multiplies the configured flow rate:

pipette.aspirate(50, source, rate=0.5)

Supported modern APIs also accept absolute flow-rate arguments:

pipette.aspirate(50, source, flow_rate=25)

Use one form per action. Absolute units are µL/s. Establish values through liquid-specific testing rather than copying another pipette's settings.

Positions

source.bottom(z=1)
source.top(z=-2)
destination.center()

• Bottom aspiration reduces residual volume but increases collision and pellet disturbance risk.
• Top or near-top dispensing can reduce contact contamination but may splash.
• Side offsets can reduce foaming but require known well geometry.
• touch_tip() can be unsafe in large wells and reservoirs; API 2.28+ rejects certain large-space uses.

Air gaps and push out

Air gaps can reduce dripping but occupy pipette capacity:

pipette.aspirate(80, source)
pipette.air_gap(10)
pipette.dispense(90, destination)

The total liquid plus air must fit the pipette. Use push_out to move the plunger a small extra amount after dispensing:

pipette.dispense(80, destination, push_out=5)

Use blowout for a larger purge. Avoid blowing into liquid when aerosols, bubbles, or cross-contamination matter.

Mixing

Standard mixing:

pipette.mix(
    repetitions=5,
    volume=40,
    location=plate["A1"].bottom(z=1),
    aspirate_flow_rate=20,
    dispense_flow_rate=30,
    final_push_out=5,
)

Choose a mix volume below the available liquid volume and pipette maximum. Account for pellets, beads, cells, foaming, and plate seals.

API 2.27 adds dynamic mixing:

well = plate["A1"]
pipette.dynamic_mix(
    aspirate_start_location=well.bottom(z=1),
    aspirate_end_location=well.bottom(z=4),
    dispense_start_location=well.bottom(z=4),
    dispense_end_location=well.bottom(z=1),
    repetitions=3,
    volume=50,
)

Dynamic movement is geometry-sensitive. Simulate, inspect the path, and dry-run with the exact labware before using it on samples.

Dynamic Aspiration and Dispensing

API 2.27 can move between two locations during one plunger action:

pipette.aspirate(
    volume=100,
    location=well.bottom(z=1),
    end_location=well.bottom(z=5),
    movement_delay=1,
)

This can follow a changing meniscus or sweep through a liquid column. It does not automatically prove that the declared liquid volume or geometry is correct.

Liquid Definitions and Meniscus

Declare setup volumes with labware-level methods:

buffer = protocol.define_liquid(
    name="Buffer",
    description="Assay buffer",
    display_color="#1F77B4",
)
reservoir.load_liquid(
    wells=["A1"],
    volume=12_000,
    liquid=buffer,
)

API 2.23 adds well.meniscus():

start_surface = reservoir["A1"].meniscus(z=-1, target="start")
end_surface = reservoir["A1"].meniscus(z=-1, target="end")

The calculated surface depends on liquid volume and labware geometry. With dynamic aspiration or dispensing, target="start" and target="end" can represent the expected surface at either end of the operation.

API 2.30 permits a start-only aspiration without an end location:

pipette.aspirate(50, reservoir["A1"].meniscus(z=-1, target="start"))

At API 2.29 and lower, that form raises an error; use the documented end-target or dynamic start/end form instead. This 2.30 fix does not add OT-2 support.

Do not rely on meniscus targeting until declared volumes, well geometry, and liquid-level behavior have been checked on the robot.

Liquid Presence Detection

Flex pressure sensors support three explicit operations:

present = pipette.detect_liquid_presence(reservoir["A1"])
pipette.require_liquid_presence(reservoir["A1"])
height = pipette.measure_liquid_height(reservoir["A1"])

Or enable a check before every aspiration:

pipette = protocol.load_instrument(
    "flex_1channel_1000",
    "left",
    tip_racks=[tips],
    liquid_presence_detection=True,
)

Operational constraints:

• Use a fresh, dry, empty tip.
• Detection can add 5–50 seconds per check depending on well depth and volume.
• An 8-channel pipette has pressure sensors only on channels 1 and 8.
• A 96-channel pipette has pressure sensors only on channels 1 and 96.
• A wet tip can defeat absence detection.
• With global detection, complex-command refills may check with a wet tip. Prefer building blocks that pick up a fresh tip immediately before every checked aspiration.
• Detection is not a substitute for source-volume planning.

Use explicit checks at critical sources when global detection would add too much time.

Partial Tip Pickup

Supported layouts:

from opentrons.protocol_api import ALL, COLUMN

pipette.configure_nozzle_layout(
    style=COLUMN,
    start="A12",
    tip_racks=[partial_rack],
)

# Partial-column operations...

pipette.configure_nozzle_layout(
    style=ALL,
    tip_racks=[full_rack],
)

Critical rules:

• configure_nozzle_layout() resets pipette.tip_racks.
• Use separate rack variables for full and partial pickup.
• Full-rack Flex 96-channel pickup requires an adapter.
• Partial Flex 96-channel pickup must not use the adapter.
• Never pass a pickup or well location that leaves active nozzles hanging outside the rack or labware.
• Deck-edge reach depends on layout and starting nozzle.
• Prefer the 96-channel pipette's column-12 nozzles for column pickup when deck reach allows.
• Simulate and perform a tip-only dry run before first physical use.

See the official Partial Tip Pickup guide for layout-specific target-well rules.

Serial Dilution Pattern

For a full 96-well plate and an 8-channel pipette:

1. Preload stock in column 1.
2. Add diluent to columns 2–12.
3. Transfer from column 1 to 2, mix, then 2 to 3, and so on.
4. Use a fresh tip set at each dilution step unless the validated method says otherwise.
5. Remove one transfer volume from column 12 if equal final volumes are needed.

The bundled template uses 13 sets of eight tips (104 individual tips), draws 8,800 µL diluent from the 12,000 µL setup volume, and collects 800 µL final liquid waste in initially empty reservoir A12. That reserve still requires validation against the source's dead volume. Waste must remain separate from diluent; the trash bin receives tips.

Referencing A-row wells addresses full columns:

pipette.transfer(
    100,
    source=plate.rows()[0][0:11],
    dest=plate.rows()[0][1:12],
    mix_after=(3, 50),
    new_tip="always",
)

Verify that the tip budget covers 11 serial steps plus diluent addition and final-volume removal.

Final Liquid-Handling Review

• Every volume is within pipette and tip range.
• Air plus liquid never exceeds capacity.
• Sources include dead volume and disposal volume.
• Destinations remain below capacity at every intermediate step.
• Mix volume is physically available.
• Positions do not contact the well bottom or pellet.
• Tip policy matches contamination boundaries.
• Multi-channel well references match the active nozzle layout.
• Liquid sensing uses fresh, dry tips.
• Simulation expansion matches the intended command order.
• Liquid-specific behavior has been checked in a dry run.