Opentrons Temperature Module

Quick Reference

Platform Compatibility

Both Opentrons Flex and OT-2

Module Generations

• GEN1 - Original temperature module
• GEN2 - Improved cooling performance, better isolation (recommended)

Key GEN2 improvements:

• Plastic insulation around plate
• Shrouds for aluminum blocks
• Better cooling when sharing deck with running Thermocycler
• Same API as GEN1

Loading the Module

from opentrons import protocol_api

metadata = {'apiLevel': '2.19'}

def run(protocol: protocol_api.ProtocolContext):
    # Load Temperature Module GEN2
    temp_mod = protocol.load_module("temperature module gen2", "D1")  # Flex
    # temp_mod = protocol.load_module("temperature module gen2", "4")  # OT-2

    # Load labware on module
    cold_plate = temp_mod.load_labware("opentrons_96_aluminumblock_generic_pcr_strip_200ul")

Module versions:

• "temperature module gen2" - GEN2 (recommended)
• "temperatureModuleV1" - GEN1 (legacy)

Deck slot: Any compatible slot (Flex: A1-D3, OT-2: 1-11)

Loading Labware

Standalone Adapters (API 2.15+)

Recommended approach - Load adapter first, then labware:

# 96-well aluminum block
temp_block = temp_mod.load_adapter("opentrons_96_well_aluminum_block")
sample_plate = temp_block.load_labware("opentrons_96_wellplate_200ul_pcr_full_skirt")

Available adapters:

Block-and-Tube Combinations

For tube racks with aluminum blocks:

# 24-well block with various tube types
tubes_nest = temp_mod.load_labware("opentrons_24_aluminumblock_nest_1.5ml_snapcap")
tubes_generic = temp_mod.load_labware("opentrons_24_aluminumblock_generic_2ml_screwcap")

Legacy Block-and-Plate Combinations

# Pre-configured combinations (older API versions)
plate_combo = temp_mod.load_labware("opentrons_96_aluminumblock_biorad_wellplate_200ul")

Recommendation: Use standalone adapters (API 2.15+) for better flexibility.

Temperature Control

Setting Temperature

# Set temperature and wait until reached
temp_mod.set_temperature(celsius=4)

# Protocol waits here until 4°C is reached

# Perform operations at target temperature
pipette.transfer(100, cold_reagent, dest_plate.wells())

Temperature range: 4-95°C (1°C resolution)

Behavior: Protocol execution blocks (waits) until target temperature is reached.

Temperature Status

# Get current temperature
current_temp = temp_mod.temperature
protocol.comment(f"Module at {current_temp}°C")

# Check status
status = temp_mod.status
# Returns: "holding at target" or "idle"

if status == "holding at target":
    protocol.comment("Temperature stable, proceeding")

Deactivating

# Turn off temperature control
temp_mod.deactivate()

Important: Module does not automatically deactivate at protocol end. Must be manually turned off via Opentrons App if protocol completes or is cancelled.

Common Patterns

Keeping Reagents Cold

# Keep enzymes and master mix cold throughout protocol
temp_mod = protocol.load_module("temperature module gen2", "D1")
reagent_block = temp_mod.load_adapter("opentrons_24_aluminumblock_nest_1.5ml_snapcap")

# Set to 4°C before starting
temp_mod.set_temperature(4)

# Reagents stay cold during entire protocol
pipette.transfer(10, reagent_block["A1"], dest_plate.wells())
# ... rest of protocol ...

# Turn off at end
temp_mod.deactivate()

Pre-Warming Reaction Components

# Pre-warm buffer to 37°C
temp_mod = protocol.load_module("temperature module gen2", "C2")
warm_block = temp_mod.load_adapter("opentrons_96_well_aluminum_block")
warm_plate = warm_block.load_labware("corning_96_wellplate_360ul_flat")

# Set to reaction temperature
temp_mod.set_temperature(37)

# Use pre-warmed components
pipette.transfer(50, warm_plate["A1"], reaction_plate.wells())

temp_mod.deactivate()

Cooling After Thermal Processing

# Cool samples after PCR or heat inactivation
temp_mod = protocol.load_module("temperature module gen2", "D3")
cooling_block = temp_mod.load_adapter("opentrons_96_well_aluminum_block")

# Set to 4°C for cooling
temp_mod.set_temperature(4)

# Move hot samples to cooling block with gripper (Flex)
protocol.move_labware(hot_plate, temp_mod, use_gripper=True)

# Hold at 4°C
protocol.delay(minutes=5)

# Continue processing cooled samples
temp_mod.deactivate()

Temperature-Sensitive Enzyme Reaction

# Restriction digest at optimal temperature
temp_mod = protocol.load_module("temperature module gen2", "D1")
rxn_block = temp_mod.load_adapter("opentrons_96_well_aluminum_block")
rxn_plate = rxn_block.load_labware("opentrons_96_wellplate_200ul_pcr_full_skirt")

# Setup reaction
pipette.transfer(20, dna_samples, rxn_plate.wells()[:8])
pipette.transfer(5, enzyme_mix, rxn_plate.wells()[:8], mix_after=(3, 15))

# Incubate at optimal temperature
temp_mod.set_temperature(37)
protocol.delay(hours=2)

# Heat inactivation
temp_mod.set_temperature(65)
protocol.delay(minutes=20)

# Cool for downstream processing
temp_mod.set_temperature(4)

temp_mod.deactivate()

Maintaining Temperature During Multi-Step Protocol

# Keep samples at 4°C except during specific steps
cold_storage = protocol.load_module("temperature module gen2", "D1")
cold_block = cold_storage.load_adapter("opentrons_96_well_aluminum_block")
sample_plate = cold_block.load_labware("biorad_96_wellplate_200ul_pcr")

# Set to 4°C at start
cold_storage.set_temperature(4)

# Samples stay cold on module
# Move to room temp for specific operations
protocol.move_labware(sample_plate, "C2", use_gripper=True)

# ... perform room-temp operations ...

# Return to cold storage
protocol.move_labware(sample_plate, cold_storage, use_gripper=True)

cold_storage.deactivate()

Integration with Other Modules

With Thermocycler

# Pre-cool samples before PCR
temp_mod = protocol.load_module("temperature module gen2", "D1")
tc_mod = protocol.load_module("thermocyclerModuleV2")

cold_block = temp_mod.load_adapter("opentrons_96_well_aluminum_block")
sample_plate = cold_block.load_labware("opentrons_96_wellplate_200ul_pcr_full_skirt")

# Keep samples cold
temp_mod.set_temperature(4)

# Setup PCR mix (samples stay cold)
# ... pipetting ...

# Transfer to thermocycler
tc_mod.open_lid()
protocol.move_labware(sample_plate, tc_mod, use_gripper=True)

# Run PCR
tc_mod.close_lid()
# ... thermal cycling ...

# Return to cold storage after PCR
tc_mod.open_lid()
protocol.move_labware(sample_plate, temp_mod, use_gripper=True)

temp_mod.deactivate()

With Heater-Shaker

# Cool after shaking/heating
hs_mod = protocol.load_module("heaterShakerModuleV1", "D1")
temp_mod = protocol.load_module("temperature module gen2", "D2")

# Pre-cool temperature module
temp_mod.set_temperature(4)

# Heat/shake on heater-shaker
hs_mod.set_and_wait_for_temperature(65)
hs_mod.close_labware_latch()
hs_mod.set_and_wait_for_shake_speed(1000)
protocol.delay(minutes=10)
hs_mod.deactivate_shaker()
hs_mod.deactivate_heater()
hs_mod.open_labware_latch()

# Transfer to temperature module for cooling
protocol.move_labware(reaction_plate, temp_mod, use_gripper=True)
protocol.delay(minutes=3)

temp_mod.deactivate()

With Magnetic Block

# Temperature-controlled magnetic separation
mag_block = protocol.load_module("magneticBlockV1", "D1")
temp_mod = protocol.load_module("temperature module gen2", "D2")

# Cool elution buffer
temp_mod.set_temperature(4)

# Magnetic separation
protocol.move_labware(sample_plate, mag_block, use_gripper=True)
protocol.delay(minutes=3)
pipette.transfer(150, sample_plate.wells(), waste.wells())

# Elute with cold buffer
protocol.move_labware(sample_plate, temp_mod, use_gripper=True)
pipette.transfer(50, cold_buffer, sample_plate.wells())

temp_mod.deactivate()

Best Practices

1. Set temperature before loading samples - Allow module to stabilize
2. Deactivate at protocol end - Prevent equipment running indefinitely
3. Use GEN2 for better performance - Improved cooling and isolation
4. Plan for thermal equilibration - Large temperature changes take time
5. Monitor ambient temperature - Affects minimum achievable temperature (4°C target requires cool room)
6. Use aluminum blocks - Better thermal contact than direct plate placement
7. Avoid pipetting during temperature changes - Wait for "holding at target" status
8. Consider thermal mass - More liquid = slower temperature equilibration
9. Don't rely on auto-deactivation - Module stays on after protocol ends
10. Check compatibility - Verify labware fits adapter/block

Common Mistakes

❌ Pipetting during temperature change:

temp_mod.set_temperature(4)
# set_temperature() blocks until reached, but safer to check status
pipette.transfer(...)  # Risk if not fully stabilized

✅ Correct:

temp_mod.set_temperature(4)
# Wait for status confirmation
if temp_mod.status == "holding at target":
    pipette.transfer(...)

❌ Not deactivating module:

temp_mod.set_temperature(4)
# ... protocol ends ...
# Module stays at 4°C indefinitely!

✅ Correct:

temp_mod.set_temperature(4)
# ... operations ...
temp_mod.deactivate()

❌ Temperature out of range:

temp_mod.set_temperature(100)  # Error: max is 95°C
temp_mod.set_temperature(0)    # Error: min is 4°C

✅ Correct:

temp_mod.set_temperature(95)  # Within range
temp_mod.set_temperature(4)   # Within range

❌ Loading labware before temperature stabilizes:

temp_mod.set_temperature(4)
# Temperature not yet reached - samples warm during cooling
pipette.transfer(100, warm_samples, temp_block.wells())

✅ Correct:

# Set temperature BEFORE loading samples
temp_mod.set_temperature(4)
# Now at 4°C, samples stay cold
pipette.transfer(100, warm_samples, temp_block.wells())

Troubleshooting

Module not reaching 4°C:

• Check ambient room temperature (module can't cool below ambient by much)
• Ensure adequate airflow around module
• Verify module is on flat, level surface
• Consider using ice packs as supplement for very cold requirements

Module not reaching high temperatures:

• Verify temperature is ≤95°C
• Check module is not in cold environment
• Allow sufficient time for large thermal mass

Temperature not stable:

• Wait for status to show "holding at target"
• Avoid high-airflow environments
• Ensure good thermal contact between labware and block
• Use appropriate adapter for labware type

Slow temperature changes:

• Normal for large temperature differences
• Use smaller labware/tubes for faster equilibration
• Pre-cool/pre-warm module before critical steps
• Consider thermal mass of samples

Module stays on after protocol:

• This is expected behavior
• Manually deactivate via Opentrons App
• Or add deactivate() to protocol end

Temperature Limits

Note: Actual minimum temperature depends on ambient conditions. In warm room (>25°C), reaching 4°C may be difficult.

Advanced Techniques

Temperature Gradient Protocol

# Ramp temperature for optimization
temp_mod = protocol.load_module("temperature module gen2", "D1")

for temp in [4, 20, 37, 55, 70]:
    temp_mod.set_temperature(temp)
    protocol.comment(f"Now at {temp}°C")

    # Perform operations at each temperature
    # ... sampling, measurements, etc. ...

    protocol.delay(minutes=5)

temp_mod.deactivate()

Dual Temperature Setup

# Maintain two different temperatures simultaneously
cold_mod = protocol.load_module("temperature module gen2", "D1")
warm_mod = protocol.load_module("temperature module gen2", "D2")

cold_mod.set_temperature(4)
warm_mod.set_temperature(37)

# Use both temperature zones in protocol
pipette.transfer(10, cold_mod.labware["A1"], reaction_plate.wells())
pipette.transfer(50, warm_mod.labware["A1"], reaction_plate.wells())

cold_mod.deactivate()
warm_mod.deactivate()

Temperature Shock Protocol

# Quick temperature changes for cell work
temp_mod = protocol.load_module("temperature module gen2", "D1")
cell_plate = temp_mod.load_labware("corning_96_wellplate_360ul_flat")

# Ice incubation
temp_mod.set_temperature(4)
protocol.delay(minutes=20)

# Heat shock
temp_mod.set_temperature(42)
protocol.delay(seconds=45)

# Recovery
temp_mod.set_temperature(37)
protocol.delay(minutes=5)

temp_mod.deactivate()

GEN2 vs GEN1

Recommendation: Use GEN2 for all new protocols.

API Version Requirements

• Minimum API version: 2.0 (temperature module support)
• Standalone adapters: API 2.15+
• Recommended: 2.19+ for full feature support

Additional Resources

• Temperature Module Documentation: https://docs.opentrons.com/v2/modules/temperature_module.html
• Labware Library: https://labware.opentrons.com/
• Opentrons Support: https://support.opentrons.com/

Related Skills

• opentrons - Main Opentrons Python API skill
• opentrons-heater-shaker - Temperature control with mixing (37-95°C)
• opentrons-thermocycler - PCR thermal cycling (4-99°C block)
• opentrons-gripper - Automated labware movement (Flex)
• opentrons-magnetic-block - Magnetic bead separation