Scientific Opentrons Integration

# Opentrons Integration

## Overview

Opentrons is a Python-based lab automation platform for Flex and OT-2 robots. Write Protocol API v2 protocols for liquid handling, control hardware modules (heater-shaker, thermocycler), manage labware, for automated pipetting workflows.

## When to Use This Skill

This skill should be used when:
- Writing Opentrons Protocol API v2 protocols in Python
- Automating liquid handling workflows on Flex or OT-2 robots
- Controlling hardware modules (temperature, magnetic, heater-shaker, thermocycler)
- Setting up labware configurations and deck layouts
- Implementing complex pipetting operations (serial dilutions, plate replication, PCR setup)
- Managing tip usage and optimizing protocol efficiency
- Working with multi-channel pipettes for 96-well plate operations
- Simulating and testing protocols before robot execution

## Core Capabilities

### 1. Protocol Structure and Metadata

Every Opentrons protocol follows a standard structure:

```python
from opentrons import protocol_api

# Metadata
metadata = {
    'protocolName': 'My Protocol',
    'author': 'Name <email@example.com>',
    'description': 'Protocol description',
    'apiLevel': '2.19'  # Use latest available API version
}

# Requirements (optional)
requirements = {
    'robotType': 'Flex',  # or 'OT-2'
    'apiLevel': '2.19'
}

# Run function
def run(protocol: protocol_api.ProtocolContext):
    # Protocol commands go here
    pass
```

**Key elements:**
- Import `protocol_api` from `opentrons`
- Define `metadata` dict with protocolName, author, description, apiLevel
- Optional `requirements` dict for robot type and API version
- Implement `run()` function receiving `ProtocolContext` as parameter
- All protocol logic goes inside the `run()` function

### 2. Loading Hardware

**Loading Instruments (Pipettes):**

```python
def run(protocol: protocol_api.ProtocolContext):
    # Load pipette on specific mount
    left_pipette = protocol.load_instrument(
        'p1000_single_flex',  # Instrument name
        'left',               # Mount: 'left' or 'right'
        tip_racks=[tip_rack]  # List of tip rack labware objects
    )
```

Common pipette names:
- Flex: `p50_single_flex`, `p1000_single_flex`, `p50_multi_flex`, `p1000_multi_flex`
- OT-2: `p20_single_gen2`, `p300_single_gen2`, `p1000_single_gen2`, `p20_multi_gen2`, `p300_multi_gen2`

**Loading Labware:**

```python
# Load labware directly on deck
plate = protocol.load_labware(
    'corning_96_wellplate_360ul_flat',  # Labware API name
    'D1',                                # Deck slot (Flex: A1-D3, OT-2: 1-11)
    label='Sample Plate'                 # Optional display label
)

# Load tip rack
tip_rack = protocol.load_labware('opentrons_flex_96_tiprack_1000ul', 'C1')

# Load labware on adapter
adapter = protocol.load_adapter('opentrons_flex_96_tiprack_adapter', 'B1')
tips = adapter.load_labware('opentrons_flex_96_tiprack_200ul')
```

**Loading Modules:**

```python
# Temperature module
temp_module = protocol.load_module('temperature module gen2', 'D3')
temp_plate = temp_module.load_labware('corning_96_wellplate_360ul_flat')

# Magnetic module
mag_module = protocol.load_module('magnetic module gen2', 'C2')
mag_plate = mag_module.load_labware('nest_96_wellplate_100ul_pcr_full_skirt')

# Heater-Shaker module
hs_module = protocol.load_module('heaterShakerModuleV1', 'D1')
hs_plate = hs_module.load_labware('corning_96_wellplate_360ul_flat')

# Thermocycler module (takes up specific slots automatically)
tc_module = protocol.load_module('thermocyclerModuleV2')
tc_plate = tc_module.load_labware('nest_96_wellplate_100ul_pcr_full_skirt')
```

### 3. Liquid Handling Operations

**Basic Operations:**

```python
# Pick up tip
pipette.pick_up_tip()

# Aspirate (draw liquid in)
pipette.aspirate(
    volume=100,           # Volume in µL
    location=source['A1'] # Well or location object
)

# Dispense (expel liquid)
pipette.dispense(
    volume=100,
    location=dest['B1']
)

# Drop tip
pipette.drop_tip()

# Return tip to rack
pipette.return_tip()
```

**Complex Operations:**

```python
# Transfer (combines pick_up, aspirate, dispense, drop_tip)
pipette.transfer(
    volume=100,
    source=source_plate['A1'],
    dest=dest_plate['B1'],
    new_tip='always'  # 'always', 'once', or 'never'
)

# Distribute (one source to multiple destinations)
pipette.distribute(
    volume=50,
    source=reservoir['A1'],
    dest=[plate['A1'], plate['A2'], plate['A3']],
    new_tip='once'
)

# Consolidate (multiple sources to one destination)
pipette.consolidate(
    volume=50,
    source=[plate['A1'], plate['A2'], plate['A3']],
    dest=reservoir['A1'],
    new_tip='once'
)
```

**Advanced Techniques:**

```python
# Mix (aspirate and dispense in same location)
pipette.mix(
    repetitions=3,
    volume=50,
    location=plate['A1']
)

# Air gap (prevent dripping)
pipette.aspirate(100, source['A1'])
pipette.air_gap(20)  # 20µL air gap
pipette.dispense(120, dest['A1'])

# Blow out (expel remaining liquid)
pipette.blow_out(location=dest['A1'].top())

# Touch tip (remove droplets on tip exterior)
pipette.touch_tip(location=plate['A1'])
```

**Flow Rate Control:**

```python
# Set flow rates (µL/s)
pipette.flow_rate.aspirate = 150
pipette.flow_rate.dispense = 300
pipette.flow_rate.blow_out = 400
```

### 4. Accessing Wells and Locations

**Well Access Methods:**

```python
# By name
well_a1 = plate['A1']

# By index
first_well = plate.wells()[0]

# All wells
all_wells = plate.wells()  # Returns list

# By rows
rows = plate.rows()  # Returns list of lists
row_a = plate.rows()[0]  # All wells in row A

# By columns
columns = plate.columns()  # Returns list of lists
column_1 = plate.columns()[0]  # All wells in column 1

# Wells by name (dictionary)
wells_dict = plate.wells_by_name()  # {'A1': Well, 'A2': Well, ...}
```

**Location Methods:**

```python
# Top of well (default: 1mm below top)
pipette.aspirate(100, well.top())
pipette.aspirate(100, well.top(z=5))  # 5mm above top

# Bottom of well (default: 1mm above bottom)
pipette.aspirate(100, well.bottom())
pipette.aspirate(100, well.bottom(z=2))  # 2mm above bottom

# Center of well
pipette.aspirate(100, well.center())
```

### 5. Hardware Module Control

**Temperature Module:**

```python
# Set temperature
temp_module.set_temperature(celsius=4)

# Wait for temperature
temp_module.await_temperature(celsius=4)

# Deactivate
temp_module.deactivate()

# Check status
current_temp = temp_module.temperature  # Current temperature
target_temp = temp_module.target  # Target temperature
```

**Magnetic Module:**

```python
# Engage (raise magnets)
mag_module.engage(height_from_base=10)  # mm from labware base

# Disengage (lower magnets)
mag_module.disengage()

# Check status
is_engaged = mag_module.status  # 'engaged' or 'disengaged'
```

**Heater-Shaker Module:**

```python
# Set temperature
hs_module.set_target_temperature(celsius=37)

# Wait for temperature
hs_module.wait_for_temperature()

# Set shake speed
hs_module.set_and_wait_for_shake_speed(rpm=500)

# Close labware latch
hs_module.close_labware_latch()

# Open labware latch
hs_module.open_labware_latch()

# Deactivate heater
hs_module.deactivate_heater()

# Deactivate shaker
hs_module.deactivate_shaker()
```

**Thermocycler Module:**

```python
# Open lid
tc_module.open_lid()

# Close lid
tc_module.close_lid()

# Set lid temperature
tc_module.set_lid_temperature(celsius=105)

# Set block temperature
tc_module.set_block_temperature(
    temperature=95,
    hold_time_seconds=30,
    hold_time_minutes=0.5,
    block_max_volume=50  # µL per well
)

# Execute profile (PCR cycling)
profile = [
    {'temperature': 95, 'hold_time_seconds': 30},
    {'temperature': 57, 'hold_time_seconds': 30},
    {'temperature': 72, 'hold_time_seconds': 60}
]
tc_module.execute_profile(
    steps=profile,
    repetitions=30,
    block_max_volume=50
)

# Deactivate
tc_module.deactivate_lid()
tc_module.deactivate_block()
```

**Absorbance Plate Reader:**

```python
# Initialize and read
result = plate_reader.read(wavelengths=[450, 650])

# Access readings
absorbance_data = result  # Dict with wavelength keys
```

### 6. Liquid Tracking and Labeling

**Define Liquids:**

```python
# Define liquid types
water = protocol.define_liquid(
    name='Water',
    description='Ultrapure water',
    display_color='#0000FF'  # Hex color code
)

sample = protocol.define_liquid(
    name='Sample',
    description='Cell lysate sample',
    display_color='#FF0000'
)
```

**Load Liquids into Wells:**

```python
# Load liquid into specific wells
reservoir['A1'].load_liquid(liquid=water, volume=50000)  # µL
plate['A1'].load_liquid(liquid=sample, volume=100)

# Mark wells as empty
plate['B1'].load_empty()
```

### 7. Protocol Control and Utilities

**Execution Control:**

```python
# Pause protocol
protocol.pause(msg='Replace tip box and resume')

# Delay
protocol.delay(seconds=60)
protocol.delay(minutes=5)

# Comment (appears in logs)
protocol.comment('Starting serial dilution')

# Home robot
protocol.home()
```

**Conditional Logic:**

```python
# Check if simulating
if protocol.is_simulating():
    protocol.comment('Running in simulation mode')
else:
    protocol.comment('Running on actual robot')
```

**Rail Lights (Flex only):**

```python
# Turn lights on
protocol.set_rail_lights(on=True)

# Turn lights off
protocol.set_rail_lights(on=False)
```

### 8. Multi-Channel and 8-Channel Pipetting

When using multi-channel pipettes:

```python
# Load 8-channel pipette
multi_pipette = protocol.load_instrument(
    'p300_multi_gen2',
    'left',
    tip_racks=[tips]
)

# Access entire column with single well reference
multi_pipette.transfer(
    volume=100,
    source=source_plate['A1'],  # Accesses entire column 1
    dest=dest_plate['A1']       # Dispenses to entire column 1
)

# Use rows() for row-wise operations
for row in plate.rows():
    multi_pipette.transfer(100, reservoir['A1'], row[0])
```

### 9. Common Protocol Patterns

**Serial Dilution:**

```python
def run(protocol: protocol_api.ProtocolContext):
    # Load labware
    tips = protocol.load_labware('opentrons_flex_96_tiprack_200ul', 'D1')
    reservoir = protocol.load_labware('nest_12_reservoir_15ml', 'D2')
    plate = protocol.load_labware('corning_96_wellplate_360ul_flat', 'D3')

    # Load pipette
    p300 = protocol.load_instrument('p300_single_flex', 'left', tip_racks=[tips])

    # Add diluent to all wells except first
    p300.transfer(100, reservoir['A1'], plate.rows()[0][1:])

    # Serial dilution across row
    p300.transfer(
        100,
        plate.rows()[0][:11],  # Source: wells 0-10
        plate.rows()[0][1:],   # Dest: wells 1-11
        mix_after=(3, 50),     # Mix 3x with 50µL after dispense
        new_tip='always'
    )
```

**Plate Replication:**

```python
def run(protocol: protocol_api.ProtocolContext):
    # Load labware
    tips = protocol.load_labware('opentrons_flex_96_tiprack_1000ul', 'C1')
    source = protocol.load_labware('corning_96_wellplate_360ul_flat', 'D1')
    dest = protocol.load_labware('corning_96_wellplate_360ul_flat', 'D2')

    # Load pipette
    p1000 = protocol.load_instrument('p1000_single_flex', 'left', tip_racks=[tips])

    # Transfer from all wells in source to dest
    p1000.transfer(
        100,
        source.wells(),
        dest.wells(),
        new_tip='always'
    )
```

**PCR Setup:**

```python
def run(protocol: protocol_api.ProtocolContext):
    # Load thermocycler
    tc_mod = protocol.load_module('thermocyclerModuleV2')
    tc_plate = tc_mod.load_labware('nest_96_wellplate_100ul_pcr_full_skirt')

    # Load tips and reagents
    tips = protocol.load_labware('opentrons_flex_96_tiprack_200ul', 'C1')
    reagents = protocol.load_labware('opentrons_24_tuberack_nest_1.5ml_snapcap', 'D1')

    # Load pipette
    p300 = protocol.load_instrument('p300_single_flex', 'left', tip_racks=[tips])

    # Open thermocycler lid
    tc_mod.open_lid()

    # Distribute master mix
    p300.distribute(
        20,
        reagents['A1'],
        tc_plate.wells(),
        new_tip='once'
    )

    # Add samples (example for first 8 wells)
    for i, well in enumerate(tc_plate.wells()[:8]):
        p300.transfer(5, reagents.wells()[i+1], well, new_tip='always')

    # Run PCR
    tc_mod.close_lid()
    tc_mod.set_lid_temperature(105)

    # PCR profile
    tc_mod.set_block_temperature(95, hold_time_seconds=180)

    profile = [
        {'temperature': 95, 'hold_time_seconds': 15},
        {'temperature': 60, 'hold_time_seconds': 30},
        {'temperature': 72, 'hold_time_seconds': 30}
    ]
    tc_mod.execute_profile(steps=profile, repetitions=35, block_max_volume=25)

    tc_mod.set_block_temperature(72, hold_time_minutes=5)
    tc_mod.set_block_temperature(4)

    tc_mod.deactivate_lid()
    tc_mod.open_lid()
```

## Best Practices

1. **Always specify API level**: Use the latest stable API version in metadata
2. **Use meaningful labels**: Label labware for easier identification in logs
3. **Check tip availability**: Ensure sufficient tips for protocol completion
4. **Add comments**: Use `protocol.comment()` for debugging and logging
5. **Simulate first**: Always test protocols in simulation before running on robot
6. **Handle errors gracefully**: Add pauses for manual intervention when needed
7. **Consider timing**: Use delays when protocols require incubation periods
8. **Track liquids**: Use liquid tracking for better setup validation
9. **Optimize tip usage**: Use `new_tip='once'` when appropriate to save tips
10. **Control flow rates**: Adjust flow rates for viscous or volatile liquids

## Troubleshooting

**Common Issues:**

- **Out of tips**: Verify tip rack capacity matches protocol requirements
- **Labware collisions**: Check deck layout for spatial conflicts
- **Volume errors**: Ensure volumes don't exceed well or pipette capacities
- **Module not responding**: Verify module is properly connected and firmware is updated
- **Inaccurate volumes**: Calibrate pipettes and check for air bubbles
- **Protocol fails in simulation**: Check API version compatibility and labware definitions

## Resources

For detailed API documentation, see `references/api_reference.md` in this skill directory.

For example protocol templates, see `scripts/` directory.