Thermocycler Module¶
The Thermocycler Module provides on-deck, fully automated thermocycling, and can heat and cool very quickly during operation. The module’s block can reach and maintain temperatures between 4 and 99 °C. The module’s lid can heat up to 110 °C.
The Thermocycler is represented in code by a ThermocyclerContext object, which has methods for controlling the lid, controlling the block, and setting profiles — timed heating and cooling routines that can be repeated automatically.
The examples in this section will use a Thermocycler Module GEN2 loaded as follows:
tc_mod = protocol.load_module(module_name="thermocyclerModuleV2")
plate = tc_mod.load_labware(name="opentrons_96_wellplate_200ul_pcr_full_skirt")
New in version 2.13.
Lid Control¶
The Thermocycler can control the position and temperature of its lid.
To change the lid position, use open_lid() and close_lid(). When the lid is open, the pipettes can access the loaded labware.
You can also control the temperature of the lid. Acceptable target temperatures are between 37 and 110 °C. Use set_lid_temperature(), which takes one parameter: the target temperature (in degrees Celsius) as an integer. For example, to set the lid to 50 °C:
tc_mod.set_lid_temperature(temperature=50)
The protocol will only proceed once the lid temperature reaches 50 °C. This is the case whether the previous temperature was lower than 50 °C (in which case the lid will actively heat) or higher than 50 °C (in which case the lid will passively cool).
You can turn off the lid heater at any time with deactivate_lid().
Note
Lid temperature is not affected by Thermocycler profiles. Therefore you should set an appropriate lid temperature to hold during your profile before executing it. See Thermocycler Profiles for more information on defining and executing profiles.
New in version 2.0.
Block Control¶
The Thermocycler can control its block temperature, including holding at a temperature and adjusting for the volume of liquid held in its loaded plate.
Temperature¶
To set the block temperature inside the Thermocycler, use set_block_temperature(). At minimum you have to specify a temperature in degrees Celsius:
tc_mod.set_block_temperature(temperature=4)
If you don’t specify any other parameters, the Thermocycler will hold this temperature until a new temperature is set, deactivate_block() is called, or the module is powered off.
New in version 2.0.
Hold Time¶
You can optionally instruct the Thermocycler to hold its block temperature for a specific amount of time. You can specify hold_time_minutes, hold_time_seconds, or both (in which case they will be added together). For example, this will set the block to 4 °C for 4 minutes and 15 seconds:
tc_mod.set_block_temperature(
temperature=4,
hold_time_minutes=4,
hold_time_seconds=15)
Note
Your protocol will not proceed to further commands while holding at a temperature. If you don’t specify a hold time, the protocol will proceed as soon as the target temperature is reached.
New in version 2.0.
Block Max Volume¶
The Thermocycler’s block temperature controller varies its behavior based on the amount of liquid in the wells of its labware. Accurately specifying the liquid volume allows the Thermocycler to more precisely control the temperature of the samples. You should set the block_max_volume parameter to the amount of liquid in the fullest well, measured in µL. If not specified, the Thermocycler will assume samples of 25 µL.
It is especially important to specify block_max_volume when holding at a temperature. For example, say you want to hold larger samples at a temperature for a short time:
tc_mod.set_block_temperature(
temperature=4,
hold_time_seconds=20,
block_max_volume=80)
If the Thermocycler assumes these samples are 25 µL, it may not cool them to 4 °C before starting the 20-second timer. In fact, with such a short hold time they may not reach 4 °C at all!
New in version 2.0.
Thermocycler Profiles¶
In addition to executing individual temperature commands, the Thermocycler can automatically cycle through a sequence of block temperatures to perform heat-sensitive reactions. These sequences are called profiles, which are defined in the Protocol API as lists of dictionaries. Each dictionary within the profile should have a temperature key, which specifies the temperature of the step, and either or both of hold_time_seconds and hold_time_minutes, which specify the duration of the step.
For example, this profile commands the Thermocycler to reach 10 °C and hold for 30 seconds, and then to reach 60 °C and hold for 45 seconds:
profile = [
{"temperature":10, "hold_time_seconds":30},
{"temperature":60, "hold_time_seconds":45}
]
Once you have written the steps of your profile, execute it with execute_profile(). This function executes your profile steps multiple times depending on the repetitions parameter. It also takes a block_max_volume parameter, which is the same as that of the set_block_temperature() function.
For instance, a PCR prep protocol might define and execute a profile like this:
profile = [
{"temperature":95, "hold_time_seconds":30},
{"temperature":57, "hold_time_seconds":30},
{"temperature":72, "hold_time_seconds":60}
]
tc_mod.execute_profile(steps=profile, repetitions=20, block_max_volume=32)
In terms of the actions that the Thermocycler performs, this would be equivalent to nesting set_block_temperature commands in a for loop:
for i in range(20):
tc_mod.set_block_temperature(95, hold_time_seconds=30, block_max_volume=32)
tc_mod.set_block_temperature(57, hold_time_seconds=30, block_max_volume=32)
tc_mod.set_block_temperature(72, hold_time_seconds=60, block_max_volume=32)
However, this code would generate 60 lines in the protocol’s run log, while executing a profile is summarized in a single line. Additionally, you can set a profile once and execute it multiple times (with different numbers of repetitions and maximum volumes, if needed).
Note
Temperature profiles only control the temperature of the block in the Thermocycler. You should set a lid temperature before executing the profile using set_lid_temperature().
New in version 2.0.
Auto-sealing Lids¶
Starting in robot software version 8.2.0, you can use the Opentrons Tough PCR Auto-sealing Lid to reduce evaporation on the Thermocycler. The auto-sealing lids are designed for automated use with the Flex Gripper, although you can move them manually if needed. They also work with the Opentrons Flex Deck Riser adapter, which keeps lids away from the unsterilized deck and provides better access for the gripper.
Use the following API load names for the auto-sealing lid and deck riser:
Labware |
API load name |
|---|---|
Opentrons Tough PCR Auto-sealing Lid |
|
Opentrons Flex Deck Riser |
|
Load the riser directly onto the deck with ProtocolContext.load_adapter(). Load the auto-sealing lid onto a compatible location (the deck, the riser, or another lid) with the appropriate load_labware() method. You can create a stack of up to five auto-sealing lids. If you try to stack more than five lids, the API will raise an error.
Setting up the riser and preparing a lid to use on the Thermocycler generally consists of the following steps:
Load the riser on the deck.
Load the lids onto the adapter.
Load or move a PCR plate onto the Thermocycler.
Move a lid onto the PCR plate.
Close the Thermocycler.
The following code sample shows how to perform these steps, using the riser and three auto-sealing lids. In a full protocol, you would likely have additional steps, such as pipetting to or from the PCR plate.
# load riser
riser = protocol.load_adapter(
load_name="opentrons_flex_deck_riser", location="A2"
)
# load three lids
lid_1 = riser.load_labware("opentrons_tough_pcr_auto_sealing_lid")
lid_2 = lid_1.load_labware("opentrons_tough_pcr_auto_sealing_lid")
lid_3 = lid_2.load_labware("opentrons_tough_pcr_auto_sealing_lid")
# load plate on Thermocycler
plate = protocol.load_labware(
load_name="opentrons_96_wellplate_200ul_pcr_full_skirt", location=tc_mod
)
# move lid to PCR plate
protocol.move_labware(labware=lid_3, new_location=plate, use_gripper=True)
# close Thermocycler
tc_mod.close_lid()
Warning
When using the auto-sealing lids, do not affix a rubber automation seal to the inside of the Thermocycler lid. The Thermocycler will not close properly.
When you’re finished with a lid, use the gripper to dispose of it in either the waste chute or a trash bin:
tc_mod.open_lid()
protocol.move_labware(labware=lid_3, new_location=trash, use_gripper=True)
New in version 2.16: TrashBin and WasteChute objects can accept lids.
You can then move the PCR plate off of the Thermocycler. The Flex Gripper can’t move a plate that has a lid on top of it. Always move the lid first, then the plate.
Changes with the GEN2 Thermocycler Module¶
All methods of ThermocyclerContext work with both the GEN1 and GEN2 Thermocycler. One practical difference is that the GEN2 module has a plate lift feature to make it easier to remove the plate manually or with the Opentrons Flex Gripper. To activate the plate lift, press the button on the Thermocycler for three seconds while the lid is open. If you need to do this in the middle of a run, call pause(), lift and move the plate, and then resume the run.