Your OT-2 moves pipettes around in 3D space. For it do that accurately and precisely, you need to calibrate it.
Positional calibration happens in four parts:
Pipette offset calibration.
Tip length calibration.
In this article, you'll learn what each part does, and how they all work together to move your pipettes to the right places.
What it does
Deck calibration makes sure your OT-2 moves its pipettes in the correct directions, and by the correct distances. It gives your OT-2 its sense of direction and its sense of scale.
It compensates for a few different kinds of slight physical variation:
Variation in how the deck is attached. The OT-2's deck is removable, and it has some wiggle room when it's attached to the OT-2's frame. It might be rotated a little bit.
Variation in the rails that the pipettes ride around on. Those rails might not be perfectly perpendicular to each other, or to the rest of the OT-2.
Variation in the motors and pulleys. A system of motors and pulleys moves the pipettes forward, back, left, and right. The amount that those motors need to turn to move the pipettes a given distance might not perfectly match the theoretical amount.
Those variations can be caused by:
Normal variation from how the factory assembles the OT-2.
Jostling from shipping or moving.
Normal variation from when you unbox and assemble the OT-2.
Mechanical shifts from using the OT-2 a lot, or over a long time.
A bad deck calibration can cause problems like the pipette being off-position by a direction or amount that changes depending on where it is on the deck.
Deck calibration doesn't compensate for the deck being translated (shifted straight forward, back, left, right, up, or down). So, on its own, it isn't enough to make sure your pipettes can accurately move to targeted locations. That gap is filled by pipette offset calibration, tip length calibration, and labware calibration.
The OT-2 also can't currently account for the deck being tilted or bent. It assumes the whole deck sits perfectly level.
How it works
Deck calibration works by measuring the relative alignment between:
The OT-2's deck, the surface that holds all your labware.
The OT-2's gantry, the structure and machinery that carries the pipettes around.
When you calibrate the deck, the Opentrons App will point out some reference points that are precision-engraved into the deck surface. You'll manually jog the pipette to each of those points.
This shows your OT-2 how it needs to move its motors in order to travel between those points. By extension, it gives your OT-2 its sense of direction and scale, relative to the deck. It will scale, rotate, and shear (but not translate) its internal coordinate system to match. 🤓
The OT-2 will use the new deck calibration until you do deck calibration again.
When and how to do it
You can choose when to calibrate the deck. The Opentrons App will guide you through it. See Get started: Calibrate the deck.
You can recalibrate the deck whenever you want, but you only need to do it once in a while. You should only need to do it:
As part of setting the OT-2 up for the first time.
After moving the OT-2 more than a short distance.
Once or twice per year, for maintenance.
When troubleshooting certain movement, positioning, and calibration problems.
Tip length calibration
What it does
When a pipette picks up a tip, the distance that the tip presses onto the pipette nozzle will vary from pipette to pipette—even between pipettes of the same exact model. (This is because of normal manufacturing variation in the pipette nozzles.)
Tip length calibration accounts for this. When you do a tip length calibration, it informs your OT-2 how that model of tip fits onto the nozzle of that specific, physical pipette.
This helps inform the OT-2 how far up or down to move that pipette when it's running your protocol so that the tip is positioned at the correct height.
To run a protocol, you need a tip length calibration for every combination of physical pipette and model of tip that's used.
Tip length calibration doesn't account for differences between individual tips. The OT-2 assumes that each tip is consistent with other tips of the same model. For example, if your protocol has a P300 Single-Channel pipette using 2 racks of 96 Opentrons 300 µL tips, each of those 192 tips will share the same tip length calibration. The OT-2 assumes that each of those 192 tips presses onto that pipette in the same way, forming the same total length.
Tip length calibration can also compensate for small differences between a tip's expected length (according to its labware definition) and its true, physical length.
But it won't let you substitute entirely different tips for each other. For example, if you want to use 3rd-party Fisherbrand 300 µL tips, don't try to trick the OT-2 by having your protocol load Opentrons 300 µL tips, physically providing the Fisherbrand 300 µL tips instead, and expecting tip length calibration to cover up the difference. Either physically provide the Opentrons 300 µL tips that the protocol expects, or rewrite your protocol to load Fisherbrand 300 µL tips with a custom labware definition. Otherwise, you might run into confusing movement, positioning, and calibration problems.
How it works
Tip length calibration measures how far the bottom of the tip extends below the bottom of the pipette nozzle.
During tip length calibration:
You'll manually jog the bare pipette—without any tip attached—so that its nozzle just barely touches a flat surface. The OT-2 will note the pipette's height.
The pipette will pick up a tip.
You'll manually jog the pipette—with the tip attached, this time—so that it just barely touches that same flat surface. The OT-2 will note the pipette's height again.
The OT-2 will take the difference between the two heights as the tip length.
The OT-2 will keep that tip length stored for any time in the future when it uses that specific pipette with that model of tip.
The OT-2 gives each physical pipette its own set of tip length calibrations. For example, if your OT-2 has two P300 Single-Channel GEN2 pipettes, each one could have one tip length calibration for Opentrons 300 µL tips and one tip length calibration for Opentrons 200 µL filter tips. And all four of those tip length calibrations would be independent from each other.
When and how to do it
The Opentrons App will automatically prompt you to calibrate tip length when you need to.
Specifically, it can prompt you:
Before running a protocol, if it uses a pipette and tip pairing for which you haven't yet done a tip length calibration.
Before pipette offset calibration. (See the "Pipette offset calibration" section, below.) Tip length calibration is a prerequisite for pipette offset calibration.
You can also manually choose to recalibrate, if you suspect something is wrong with your current tip length calibration. To do so, go to your OT-2's Pipettes & modules page.
Pipette offset calibration
What it does
When the OT-2 moves a pipette to different points, there might be a systematic error. For example, your particular P300 Single-Channel might always go 1 mm too far to the right when it's attached to your OT-2's left mount.
Pipette offset calibration compensates for this. It nudges the pipette—wherever it goes—by some constant amount to make the true tip position match what the protocol targeted. The offset can be forward, back, left, right, up, or down.
This accounts for:
Manufacturing variation in the pipette.
Manufacturing variation in the mount.
Variation in how that pipette attaches to that mount.
How the whole deck is shifted side-to-side. (On the other hand, how the deck is rotated is one of the things that deck calibration accounts for.)
How it works
When you calibrate pipette offset:
The OT-2 will attempt to move the pipette to a predefined reference point.
You'll manually adjust the pipette so that it's exactly centered over it.
The OT-2 will save that adjustment and use it until you overwrite it.
Your OT-2 has one pipette offset calibration per physical pipette, per mount. For example, if you have a P300 Single-Channel GEN2 pipette, it will have separate pipette offset calibrations for when you attach it to the left mount and when you attach it to the right mount. And if you get a second P300 Single-Channel GEN2, it will have its own set of pipette offset calibrations.
When and how to do it
The Opentrons App will automatically prompt you to calibrate pipette offset when you need to.
Specifically, it can prompt you:
After attaching a pipette, if you've never calibrated pipette offset for that pipette on that mount before.
Before running a protocol, if one of the pipettes that it uses doesn't already have a pipette offset calibration for that mount, for whatever reason.
After recalibrating tip length, if that model of tip was the one used when you last did pipette offset calibration for that pipette on that mount. See the "Calibration dependencies and invalidations" section, below.
If you suspect there's something wrong with your existing pipette offset calibration, you can also start a pipette offset calibration yourself. Go to your OT-2's Pipettes & modules page and select Calibrate offset.
For details on the process itself, see Get started: Calibrate tip length and pipette offset.
What labware calibration does
Labware calibration fine-tunes the position of labware on the deck.
Theoretically, since the OT-2 knows the labware's geometry and where each slot on the deck is, it should be able to perfectly predict the location of each well.
However, in reality, there can be slight variations in physical labware and how those labware fit into slots. Those slight variations do add up, and can cause the pipette tip to miss wells or crash. Labware calibration compensates for that.
How labware calibration works
The Opentrons App will prompt you to calibrate labware after you upload a protocol, before you run it. See Get started: Pipette and labware calibration.
For each labware in your protocol:
The OT-2 will move the pipette tip to where it thinks the top of well A1 is.
You'll manually jog the pipette to center it perfectly at the top of well A1, adjusting it forward, back, left, right, up, or down, as needed.
The OT-2 will save that adjustment for when it runs your protocol.
Then, during your protocol, any time the pipette moves to any well in that labware, the OT-2 will apply the adjustment that you made.
How do I calibrate on a well other than A1?
There is no need to do this. The OT-2 already accounts for height differences between non-identical labware by referencing each labware’s specific geometry relative to the deck slot on the OT-2.
For example, if you are using different sized tubes in the same tube rack but are noticing labware-specific calibration issues, check your labware definitions to ensure they match the physical labware you are using in your protocol.
Relationships between calibrations
The different parts of your OT-2's positional calibration—deck calibration, tip length calibration, pipette offset calibration, and labware calibration—can all be calibrated, reset, and recalibrated individually.
However, some parts depend on others. There's an order of operations to them: you might need to do one part before you can do another. And recalibrating one part can sometimes mean you also have to recalibrate other parts.
Normally, you don't need to worry about the details. The Opentrons App will automatically ensure that you calibrate things in the right order. But if you're curious, understanding the details can help you understand more about how the different parts of calibration fit together.
Running a protocol requires every calibration
Each part of positional calibration on the OT-2 is mandatory. Before you can run a protocol, you need:
A deck calibration, for the whole OT-2. You won't be able to upload a protocol without one.
A pipette offset calibration for each pipette that your protocol uses, on its respective mount. If you don't have one, the Opentrons App will prompt you to perform one after you upload the protocol.
A tip length calibration for every combination of tip and pipette that your protocol uses. If you don't have one, the Opentrons App will prompt you to perform one after you upload the protocol.
A labware calibration for each model of labware that your protocol uses. If you don't have one, the Opentrons App will prompt you to perform one after you upload the protocol.
Pipette offset calibration requires the OT-2 to have a deck calibration
When you calibrate pipette offset, you center the pipette over one of the deck's engraved reference points. Therefore, pipette offset calibration depends on the OT-2 already having a good deck calibration.
The Opentrons App won't let you start a pipette offset calibration unless the OT-2 already has a deck calibration that seems sensible, according to some basic checks.
If you recalibrate the deck later on, that will clear all your pipette offset calibrations, since they wouldn't make sense with the new deck calibration, anyway.
Pipette offset calibration requires that pipette to have one tip length calibration
The process of pipette offset calibration happens to involve picking up an Opentrons tip and using that as a pointer when centering the pipette over one of the deck's engraved reference points, instead of using the bare pipette nozzle itself. This is necessary because the pipette can't always lower far enough for its nozzle to reach the deck's surface, and, even if it could, it would be hard to tell when it's centered.
Therefore, pipette offset calibration depends on having a good tip length calibration for that model of Opentrons tip. The OT-2 needs to know how much length that tip adds to that pipette in order to set that pipette's z-offset (height adjustment).
If you try to calibrate that pipette's offset without it already having a tip length calibration for that model of tip, the Opentrons App will automatically guide you through completing that tip length calibration first.
If you redo that tip length calibration, your pipette offset calibration will be cleared, and you'll immediately be prompted to redo it. After all, the original pipette offset was only good assuming the original tip length was good—so if you're redoing that tip length calibration, it would make sense to redo that pipette offset calibration, too.
For other kinds of tips that your protocol uses—whether they're different models of Opentrons tips, or 3rd-party tips that you're using with a custom labware definition—the tip length calibrations are totally independent from pipette offset calibration.