Tip length and pipette offset calibration of OT-2 pipetting workstation
Hello everyone, I am Ethan, the head of technical support at Opentrons. This video series is the sixth installment of the 0T-2 series from unboxing to protocol operation. In the last video, we completed the deck calibration. Next, let's start the tip length calibration on the OT-2. In the tip length and pipette offset calibration operation, I need to use a calibration aluminum block. I The tips used are: 20ul and 1000ul models. I need to use a computer and OT-2, the pipette has been connected to OT2. This step has been completed in the previous video. Tip length calibration explains how the tip adapts to the nozzle of a specific physical pipette. As long as the calibration is completed, OT-2 can Know how to move the pipette up, down, and far while the protocol is running to keep the tip at the correct height. Tip length calibration does not take into account differences between individual tips, OT-2 Assuming each tip is of the same length as other tips of the same model, I will use the Devices app and the Opentrons app to initiate calibration.
First, select the device I want to calibrate. As long as I have not completed the calibration, there will be a prompt here to remind me to complete the pipette offset calibration or tip connection calibration. Now I click Calibrate. When you first start to calibrate the tip length and pipette offset, the device will ask you if you need to use a calibration aluminum block. If you do not have a calibration aluminum block, you can use the garbage chute instead. I Now there's this aluminum block, so I'm going to calibrate it with the aluminum block, and I'm going to click remember my selection for next time so it doesn't prompt next time.
I'm going to continue using the calibration block, the OT-2 will now be back to the starting position and ready for calibration, I'm going to need a tip holder, preferably from 0pentrons, and a calibration aluminum block, I've got that set up, let's get started calibration. Opentrons The app will guide me through the calibration tip length calibration. I need to place the calibration aluminum block and tip holder on the corresponding plate position. The pipette Z-axis moves to the calibration block to measure the nozzle. I need to move the pipette downwards. to ensure that the distance between the nozzle and the calibration block is less than 1 mm, at this time the nozzle is less than 1 mm from the calibration block, I will save this calibration result, the pipette will move to the first tip position in the tip rack to pick up the tip, I need to put the pipette down to Make sure the pipette spray is aligned as shown in the APP animation.
The nozzle is aligned and I want the pipette to pick up the tip, my pipette is able to pick up the tip, click the move to measure tip length button and the pipette will move back to the calibration block to measure the tip length, I need to move the pipette on a small stand to make sure It's less than 1 mm from the calibration block and the nozzle is 1 mm from the calibration block and now I'm going to save the tip length settings and I'm going to remove the calibration block to continue with pipette offset calibration, pipette offset compensation The distance between two pipettes, based on Manufacturing differences between the OT-2 and the pipettes, each pipette has its own pipette offset that I need to calibrate to ensure the highest accuracy the OT-2 already has the tip attached so it will be used for calibration process, the app walked me through the calibration of each pipette. The process of pipette calibration is similar to deck calibration, first move to slot 5 - to measure the Z channel, then I will move it so that the pipette is less than 0.1mm from the deck and the z-axis position is less than 1mm from the deck, I will save this calibration and the pipette will move to the cross in plate position 1.
Similar to the deck calibration, I need to align the pipette so that the end of the tip is in the middle of the cross in slot 1, mine was a little off at first, slowly move it so it's aligned, I align the pipette with the cross , the calibration can now be saved and the calibration is complete when the "green hook" and pipette offset complete messages appear. I'll let the pipette put the tip back into the tip holder and then move on to the next calibration, the OT-2 is now reset, and here we have the pipette length calibration and offset calibration for the P20 pipette on the left stand, I am now going to perform the same process with a P1000 pipette. First, I'm going to take the 20 μl tip rack off the deck and set it aside, and I'm going to start the tip length calibration and the pipetting offset calibration on the right side now. When I enter the device page, I can see that since I have not calibrated the pipetting holder on the right before, there is a prompt saying that I need to calibrate it now. I click Calibrate now, and the application will guide me how to adjust the tip length and pipetting holder. Pipette offset calibration, like last time I need the tip holder associated with the pipette, in this case it is 1000 μl Tip holder, I need the calibration aluminum block, I have all of these, this time the calibration starts, I need to place the calibration block in plate position 1, on the right side. Then I need to place the tip rack in plate position 8. As before, remove the top tip rack in plate position 8 and place the calibration aluminum block in plate position 1. We can now confirm the placement and continue the calibration. The P1000 pipette will be lowered onto the calibrated aluminum block, just like in the previous step I need to move it down slowly so that the nozzle is close to but not touching the calibrated aluminum block, the pipette nozzle is less than 0.1 from the calibrated aluminum block mm distance, I'm going to save this setting and then go ahead and pick up a tip, I now need to position the pipette so that it's in the center of tip A1, the nozzle of the pipette is now in the center of tip A1, and Flush with the top of the tip, I now want the pipette to pick up that tip, and the app asked me if the pipette was able to pick up the tip, which it did, click OK, and then start measuring the tip length.
We will move to the calibration block in plate position 1 and move slowly so that the tip is close to but not touching the calibration aluminum block, perfect! It now rests within 0.1mm of the calibrated aluminum block, I will now save this length, now that the tip length is calibrated and there is a "green hook" mark on the app, I now need to remove the calibration from the deck aluminum block and move on to the pipette offset calibration, so I'll choose to move on to the next step, just like the deck calibration and the pipette offset calibration before, I need to lower the pipette so that it's away from the plate Less than 5 decks 0.1 mm, the pipette tip is already infinitely close to the deck of plate No. 5, and the distance is less than 0.1 mm. I'll save this calibration and the pipette will move to the edge of the first plate, and I now need to click to move the robot until the tip is exactly in the center of the cross in the first plate. Now that the tip is exactly on the cross of the first plate position, save the calibration and I know the pipette offset calibration is complete because there is a "green hook" on the screen. I now let the pipette return the tip to the tip rack and exit calibration. OT-2 reset, tip length and pipette offset calibration completed. Before running the protocol, this concludes our video. Thanks for watching the sixth installment of this series. Now that I have calibrated the pipette length and pipette offset, we will continue with instructions on how to run the protocol. Thanks for watching, see you next time!
