Overview
Automated preparation of PCR and qPCR reactions is the single most popular application on the Opentrons platform, needed and used by hundreds of scientists worldwide. This workhorse protocol provides valuable data, but requires repetitive and precise liquid-handling to set up, making it a perfect process for automating in the biology lab. The most effective way to automate this process is for the OT-2 to combine samples and PCR mastermix in the reaction plate that will go into the thermocycler or qPCR machine. The mastermix should be prepared prior to this process, either by hand or by also using the OT-2 robot. More details on this process are below.
This workflow can be used for the following applications:
- Gene qPCR)
- NGenotyping (detection)
- Amplification for cloning
- Medical, forensic, and applied sciences
Benefits
Automation leads to several benefits such as:
Workflow
PCR preparation on the OT-2 allows for a simplified workstream that can accommodate low and high throughput PCR experiments. Examples of precise, high yield PCR experiments are shown in the technical note below, along with important parameters for efficiently adapting PCR experiments on the OT-2. The most effective way to automate this process is for the OT-2 to combine samples and PCR mastermix in the reaction plate so they can go into the thermocycler or qPCR machine. The mastermix should be prepared prior to this process, either by hand or by also using the OT-2 robot. More details on this process are below.
Instructions
STEP 1DESIGN YOUR PROTOCOL
Start from scratch with the Protocol Designer, or use a Protocol Library template
STEP 2PLACE REAGENTS ONTO ROBOT
Put premade PCR master mix on robot - or have the OT-2 make it for you. Combine master mix with forward and reverse primers.
STEP 3CALIBRATE & ACTIVATE PROTOCOL
Confirm pipetting locations & hit "run" (takes < 5 minutes)
STEP 4LOAD THERMOCYCLER & RUN
After the OT-2 has prepped all your reactions, just run your PCR machine
Notes
Reagents
The use of mastermixes that contain many components of PCR, including polymerase, dNTPs and buffer, reduce the run time and pipetting steps. A ‘super-mastermix’ that includes the mastermix along with the forward and reverse primers minimizes pipetting steps, requiring only one tip per sample. We recommend this streamlined approach for setting up PCR on the OT-2. Samples in PCR strips and 96-well PCR plates can be in a pre-cooled aluminum block. Reagents can be kept at 4ºC on the Temperature Module while running the protocol on the OT-2.
Labware
The OT-2 supports various labware for PCR. We recommend full skirted 96-well PCR plates for easy and secure placement on the deck. Due to the variety of dimensions across PCR plates we encourage users to find a labware definition that closely matches the labware you already have. In addition, several examples of PCR labware defined in our software are illustrated in Figure 1. Alternatively, the ‘labware.create’ command can be used to input the exact dimensions of your PCR plate. Refer to the Labware Library for more details about supported and custom labware on the OT-2.
Pipettes
The p10, p50 and p300 pipettes in single and multi-channel formats can accommodate total reaction volumes of PCR experiments from 10 to 200 μL. For more information on our pipettes and pipetting volumes as low as 1 μL refer to our
Author: Opentrons
Part 1 of 2: Master Mix Assembly Links: Part 1: Master Mix Assembly Part 2: Master Mix Distribution and DNA Transfer This protocol allows your robot to create a master mix solution using any reagents stored in a 2 mL Eppendorf tube rack or a 2 mL screwcap tube rack. The master mix will be created in well A1 of the trough. The ingredient information will be provided as a CSV file. See Additional Notes for more details. You will need: 4-in-1 Tube Rack Set 12-well Trough
Author: Opentrons
Part 2 of 2: Master Mix Distribution and DNA Transfer Links: Part 1: Master Mix Assembly Part 2: Master Mix Distribution and DNA Transfer This protocol allows your robot to distribute a master mix solution from well A1 of a trough to PCR strips. Robot will then transfer DNA samples to the master mix solution. You will need: 12-well Trough 8-well PCR Strips
Author: Opentrons
Purification and normalization of PCR products using a 96 well wplate kit. PCR products are added to the plate and concentration is normalized. Then products can be eluted and are concentrated in a final tube at the end.
Author: Opentrons | Partner: Medicinal Genomics
Performs setup for youPCR. Make master mixes: add 100uL of LAMP buffer, 2uL of primer, 60uL of water for each row (up to 8 rows) Distribute master mix across 96-well-flat plate Transfer samples from deep plate to the flat plate containing master mix
Varies depending on your settings
Author: Opentrons
Generate and download a custom PCR protocol by following the setup steps below, tailored to your needs.
Overview
Automated preparation of PCR and qPCR reactions is the single most popular application on the Opentrons platform, needed and used by hundreds of scientists worldwide. This workhorse protocol provides valuable data, but requires repetitive and precise liquid-handling to set up, making it a perfect process for automating in the biology lab. The most effective way to automate this process is for the OT-2 to combine samples and PCR mastermix in the reaction plate that will go into the thermocycler or qPCR machine. The mastermix should be prepared prior to this process, either by hand or by also using the OT-2 robot. More details on this process are below.
This workflow can be used for the following applications:
Benefits
Automation leads to several benefits such as:
Increased data consistency and reliability with precise pipetting and fewer pipetting steps
Increased walkaway time by preparing hundreds of samples simultaneously in a single run
Workflow
PCR preparation on the OT-2 allows for a simplified workstream that can accommodate low and high throughput PCR experiments. Examples of precise, high yield PCR experiments are shown in the technical note below, along with important parameters for efficiently adapting PCR experiments on the OT-2. The most effective way to automate this process is for the OT-2 to combine samples and PCR mastermix in the reaction plate so they can go into the thermocycler or qPCR machine. The mastermix should be prepared prior to this process, either by hand or by also using the OT-2 robot. More details on this process are below.
Instructions
STEP 1DESIGN YOUR PROTOCOL
Start from scratch with the Protocol Designer, or use a Protocol Library template
STEP 2PLACE REAGENTS ONTO ROBOT
Put premade PCR master mix on robot - or have the OT-2 make it for you. Combine master mix with forward and reverse primers.
STEP 3CALIBRATE & ACTIVATE PROTOCOL
Confirm pipetting locations & hit "run" (takes < 5 minutes)
STEP 4LOAD THERMOCYCLER & RUN
After the OT-2 has prepped all your reactions, just run your PCR machine
Notes
Reagents
The use of mastermixes that contain many components of PCR, including polymerase, dNTPs and buffer, reduce the run time and pipetting steps. A ‘super-mastermix’ that includes the mastermix along with the forward and reverse primers minimizes pipetting steps, requiring only one tip per sample. We recommend this streamlined approach for setting up PCR on the OT-2. Samples in PCR strips and 96-well PCR plates can be in a pre-cooled aluminum block. Reagents can be kept at 4ºC on the Temperature Module while running the protocol on the OT-2.
Labware
The OT-2 supports various labware for PCR. We recommend full skirted 96-well PCR plates for easy and secure placement on the deck. Due to the variety of dimensions across PCR plates we encourage users to find a labware definition that closely matches the labware you already have. In addition, several examples of PCR labware defined in our software are illustrated in Figure 1. Alternatively, the ‘labware.create’ command can be used to input the exact dimensions of your PCR plate. Refer to the Labware Library for more details about supported and custom labware on the OT-2.
Pipettes
The p10, p50 and p300 pipettes in single and multi-channel formats can accommodate total reaction volumes of PCR experiments from 10 to 200 μL. For more information on our pipettes and pipetting volumes as low as 1 μL refer to our
OT-2 PCR Prep 1/2: Master Mix Assembly
Author: Opentrons
Part 1 of 2: Master Mix Assembly Links: Part 1: Master Mix Assembly Part 2: Master Mix Distribution and DNA Transfer This protocol allows your robot to create a master mix solution using any reagents stored in a 2 mL Eppendorf tube rack or a 2 mL screwcap tube rack. The master mix will be created in well A1 of the trough. The ingredient information will be provided as a CSV file. See Additional Notes for more details. You will need: 4-in-1 Tube Rack Set 12-well Trough
OT-2 PCR Prep 2/2: Master Mix Distribution and DNA Transfer
Author: Opentrons
Part 2 of 2: Master Mix Distribution and DNA Transfer Links: Part 1: Master Mix Assembly Part 2: Master Mix Distribution and DNA Transfer This protocol allows your robot to distribute a master mix solution from well A1 of a trough to PCR strips. Robot will then transfer DNA samples to the master mix solution. You will need: 12-well Trough 8-well PCR Strips
SequalPrep™ Normalization Plate (96) Kit
Author: Opentrons
Purification and normalization of PCR products using a 96 well wplate kit. PCR products are added to the plate and concentration is normalized. Then products can be eluted and are concentrated in a final tube at the end.
youPCR Setup
Author: Opentrons | Partner: Medicinal Genomics
Performs setup for youPCR. Make master mixes: add 100uL of LAMP buffer, 2uL of primer, 60uL of water for each row (up to 8 rows) Distribute master mix across 96-well-flat plate Transfer samples from deep plate to the flat plate containing master mix
Custom PCR Setup
Varies depending on your settings
Author: Opentrons
Generate and download a custom PCR protocol by following the setup steps below, tailored to your needs.