Oct 20, 2025
Version 2
OT-2 Modular Cloning Construct Assembly V.2
- Ana Mariya Anhel1,
- Laura Cutugno1,
- Ángel oñi-Moreno2
- 1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain;
- 2Centro Nacional de Biotecnología (CNB-CSIC)
- Ángel oñi-Moreno: [email protected];
Protocol Citation: Ana Mariya Anhel, Laura Cutugno, Ángel oñi-Moreno 2025. OT-2 Modular Cloning Construct Assembly . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8p82rg2w/v2Version created by Biocomputation Lab
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 08, 2024
Last Modified: October 20, 2025
Protocol Integer ID: 103008
Keywords: modular cloning construct assembly, modular cloning construct, modular cloning, modular cloning constructs from different part, golden standard modular cloning of level, assembly of the construct, description of the lap repository entry lap, ot2, assembly, protocol in the lap entry link, version of the protocol, set of instruction, plasmid, lap repository, lap repository entry lap, protocol setup, lap format script, protocol, description of robot, temperature profile, laboratory, names of the construct
Funders Acknowledgements:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555, 2019-T1/BIO-14053
MCIN/AEI
Grant ID: CEX2020-000999-S, PID2020-117205GA-I00
European Research Council
Grant ID: 101044360
Abstract
This protocol is meant to create modular cloning constructs from different parts into a final plate and, optionally, perform the temperature profile needed to the assembly of the constructs.
The output of running this script will be the final plate(s) with the constructs and the mix needed to perform that assembly, and the corresponding map(s) with the names of the constructs in their corrresponding well which will be given by the user in the input file.
This protocol is run by using a LAP format script and its corresponding .xlsx file were different customizable variables such as the number of final combinations, volumes of transfer, type of plates, etc...
Specifically, this protocol provides a set of instructions or description of the LAP repository entry LAP-MoCloAssembly-OT2-2.0.0. You can find the script and complementary information for this specific version of the protocol in the LAP entry link and GitHub link to LAP entry documents
In our laboratory, this protocol has been used to perform plasmids using the Golden Standard modular cloning of levels 1 and level 2
The current version incorporates the following modifications:
- Description of robot and protocol setup in a separate protocols.io entry (Setting and Customizing OT-2 for LAP Entries)
- New variables added: Position Distribute Water, Touch Tip After Distributing Water, Change Tip in Water Distribution, Position Distribute Reaction Mix, Touch Tip After Distributing Reaction Mix, Change Tip in Mix Distribution, Position Distribute Acceptor/Module, Touch Tip After Distributing Acceptor/Module, Change Tip in Acceptor/Module Distribution, Max Volume Per Mix Tube In Shaker
- Iteration through all the DNA parts, first distributing acceptors and then modules, to all the final combinations that include those parts.
- Tips are being replaced every time volume is aspirated from the second reagent onwards when transferred to the mix tubes, avoiding contamination of the original reagents.
Guidelines
This protocol was developed with python 3.7.1, OT App Software Version 7.0.2 and API level version 2.14 in a Linux 4.14.74 system (these are the OT-2 specifications).
In the script several packages are used: pandas (0.25.3), openpyxl (3.1.2), math, random and numpy (1.15.1)
This protocol has been tested by assembling level 1 and level 2 constructs with parts of the golden standard database
Materials
Software
- Python 3.7.1
- opentrons software version 7.0.2
- python packages: pandas (0.25.3), numpy (1.15.1), openpyxl (3.1.2), math, random
- OT App
- Excel
OT-2 Labware
- Opentrons Tip racks
Equipment
Opentrons 96 Tip Rack 300 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Opentrons 96 Tip Rack 20 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
- PCR skirted plate
Equipment
PCR 96-plate low profile Thermo Scientific
NAME
PCR Plate, 96-well, low profile
TYPE
Thermo Scientific
BRAND
AB0800R
SKU
LINK
- Opentrons Eppendord Tube Rack
Equipment
Opentrons 24 Tube Rack with Eppendorf 1.5 mL Safe-Lock Snapcap
NAME
Tube Rack
TYPE
Opentrons
BRAND
opentrons_24_tuberack_eppendorf_1.5ml_safelock_sna
SKU
LINK
- Tube Rack Eppendorf for Heater-Shaker + 24 eppendorf tube holder
TubeHolder_Eppendorfs_HS_OT2.stl Equipment
24 Eppendorf Tube holder
NAME
Tube holder
TYPE
Opentrons
BRAND
999-00030
SKU
LINK
- 1.5mL eppendorfs (without the cap) + 4ºC cold-block with adaptor (file attach)
Equipment
BRAND™ Centrifuge Tube Mini-Cooler
NAME
ColdBlock
TYPE
BRAND
BRAND
10141921
SKU
LINK
adaptor_OT_coldblock.stl Reactives:
- Water: MilliQ water
- T4 Ligase and Ligase Buffer: T4 DNA Ligase, 100uPromegaCatalog #M1801
- Restriction Enzyme: BpiI (BbsI) (10 U/µL)Thermo FisherCatalog #ER1012 BsaI-HFv2New England BiolabsCatalog # R3733S
Equipment:
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Equipment
Single Channel Electronic Pipette (GEN2) 300uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Single Channel Electronic Pipette (GEN2) 20uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Opentrons Thermocycler Module
NAME
Thermocycler
TYPE
Opentons
BRAND
999-00174
SKU
LINK
Equipment
Opentrons Heater-Shaker Module
NAME
Heater-Shaker
TYPE
Opentrons
BRAND
999-00157
SKU
LINK
Safety warnings
If you are using the heater-shaker take in account that there is a limit of RPM that it can shake before the liquid of the eppendorfs get out. As well, take in account that there are constrictions that could prevent some labware to be placed.
This speed depends on the liquid consistency and volume
Before start
Being a one-pot reaction in this protocol only 1 restriction enzyme is used so one type of level, in the case of the golden standard database, can be produced in only 1 run
Files Preparation
Preparing Customized Template
Preparing the template (a .xlsx) with the specific variables for each experiment.
Here there is attached a template of the variable file with several sheets and a PDF file explaining each variable:
- GeneralVariables: variables related mainly to the labware that is going to be used
- PerPlateVariables: variables related to the specifications of each source plate
- PipetteVariables: variables related to the pipettes that are going to be used
- ReactionVariables: variables that will determine the final mix of the wells
- ModuleVariables: variables related to the modules used in the protocol, the thermocycler and the heater-shaker
- Combinations: set of combinations that are going to be created in the final wells, one combination per row and one DNA part per cell
- Map DNA Parts Sheet(s): sheet(s) with the names of each DNA part that can be used to create final assemblies denoted in the combinations sheet. The sheet(s) need to have also dthe name of the rows and columns of the plate and the wells that does not have any sample need to be left empty --> not included in the template but needed to be included and have the same names as established in the variable Name Map DNA Parts from the PerPlateVariables Sheet
- TemperatureProfile (Optional): a profile that will be performed in the thermocycler if set as True in the ModuleVariables sheet
TemplateVariablesMoCloAssembly.xlsx 
MoCloAssemblyInstructions_v200.pdf Note
Fill the template with the corresponding values
Store it with the name VariablesMoCloAssembly.xlsx
Note
The file should be spelt exactly VariablesMoCloAssembly.xlsx or the Python script won't work correctly
Setting the robot
Prepare the system of the robot to run the protocol
For this protocol to work we need to transfer the VariablesMoCloAssembly.xlsx to the directory /data/user_storage of the OT system that we will use to perform the protocol
As well, if we are using custom labware we need to upload it to the OT App and send it to the directory /data/labware/v2/custom_definitions/custom_beta if the labware is not there yet.
Finally, we need to make sure the package openpyxl is installed in the robot system
We can do this entire step by following the protocol Setting and Customizing OT-2 for LAP Entries with the specifications given in the text above
Running Protocol
Load script in OT-App
Now that we have transferred the variable files to the robot, we can load the script and run it in the selected robot
Note
This whole step has been developed with version 6.3.1 of the OT-App and has been tested until version 7.0.2
Indications may vary from version to version
Software
Opentrons App
NAME
Windows >=10, Mac >=10 , Ubuntu >=12.04
OS
Opentrons
DEVELOPER
REPOSITORY
Load the script in the App
Protocols -> Import -> Drag Python script
This version of the protocol was developed when the last version available of LAP-MoCloAssembly-OT2 was the 2.0.0 which script you can find attached
ScriptMoCloConstructAssembly_v200.py Note
The last script version can be found at the Github entries directory or web repository section of the LAP repository.
The name of the directory or entry should be LAP-MoCloAssembly-OT2 followed by the version.
Note
The App with version 7.0.2 analyzes your protocol before setting a robot to run, so the labware will not be shown before assigning the protocol to a specific robot when you import it into the App
Select Robot to Perform Script
Click in the protocol -> Start setup -> Choose the OT where the file VariablesMoCloAssembly.xlsx is -> Proceed To Setup
After clicking on Proceed to Setup, you should obtain, if there is no error, the positions of the labware in the Labware tab and the reagents, with their corresponding volume in the Liquids tab.
In case the protocol with the set variables cannot run, an error will be raised by the app. Many errors are contemplated already and have a specific message that will give the user a hint of what could have gone wrong.
Note
Both the volume of the reagents and the DNA parts are exactly what is needed, so it is suggested to pour always more to take in account the error of pipetting
Note
It is recommended that you perform a labware position check.
You can do it with test plates and tube racks after loading the script but before cleaning the surface. That way, you reduce the probability of contamination (using the test plates and labware) and pipetting errors (position check).
Run Protocol in OT
Make sure the needed calibrations are done
Pipettes, tip racks and tip length calibrations need to be done for the items used in this run
Labware position check is performed (if needed)
Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces
Note
Check the Opentrons page https://support.opentrons.com/s/article/Cleaning-your-OT-2? for more information about cleaning the OT-2 robot with the proper materials.
Set the labware and reagents as shown in the OT-App
Start Run
The procedure that the robot is going to do is mainly divided into 9 parts:
- (Optional) The block temperature from the thermocycler reaches a temperature set by the user in case that variable is not empty
- Distribute the needed water to each final well. The volume of water is calculated by the OT-2 according to the volume set in the variable Volume Final Each Reaction (uL) taking in account the volume of the other reagents and the number of DNA parts that is going to that specific well.
- Creation of mix(es) transferring ligases, buffer, serum and restriction enzyme to new tube(s)
- Mixing with either a pipette or heater-shaker
- Distribute mix to final plate(s)
- Distribute DNA parts to the corresponding wells
- Generate identity maps to be exported
- (Optional) Pause the program for user to put lids on the plate located in the thermocycler (if set like that in the input variable)
- (Optional) Temperature profile with thermocycler module
- (Optional) Block of thermocycler stay to the set temperature
Expected result
One or more plates where there is a mix between the different DNA parts and the mix of reagents needed to perform the MoClo assembly process, each combination will be in one well.
A sheet for every final plate will be created as well in an Excel file with the given name in the sheet GeneralVariables in the variable "Name File Final Constructs" followed by the extension .xlsx in the folder /data/user_storage of the robot where we run the script.
After-Running
Retrieve labware from the OT
Importing map from robot
There will be a map with the name set in the variable Name File Final Constructs in the sheet GeneralVariables followed by the extension .xlsx: [Name File Final Constructs].xlsx
This file will be located in the directory /data/user_storage in the robot where the script has been run.
To retrieve the file, we can Linked protocol and reproduce it by transferring the files from the robot to the computer.
Take into account that files overwrite, so if you give the final map the same name in two consecutive runs, you will get only the results of the last run.
Example
6h 6m 10s
We want to make 47 constructs that are all level 1 from 38 parts, some created in the lab, some from the golden standard database.
Citation
LINK
We are going to use the heater-shaker module to mix the assembly mix (enzyme, ligase, serum and buffer) and use a thermocycler module to perform the construct assembly with a provided temperature program.
Prepare variable file
Excel temple filled and saved with the name VariablesMoCloAssembly.xlsx
VariablesMoCloAssembly.xlsx 10m
Upload custom labware to app
We are using 2 custom labware called 3dprinted_opentrons_shaker_1.5mleppendorf and coolblocknew_12_tuberack_1500ul that have been created with the labware creator that opentrons offers (https://labware.opentrons.com/create/)
3dprinted_opentrons_shaker_1.5mleppendorf.json 
coolblocknew_12_tuberack_1500ul.json We upload it to the opentrons app (make sure that it is in the robot app) and the robot system as stated in the protocol in step Setting and Customizing OT-2 for LAP Entries
3dprinted_opentrons_shaker_1.5mleppendorf.zip 
coolblocknew_12_tuberack_1500ul.zip (these are zip files because they needed to be compressed to upload to protocols.io, but what needs to be transferred to the robot is the folder inside of the zip file)
4m
Export the variable file to the /data/user_storage folder in the robot
Command line window with scp commands to transfer the variables .xlsx from our computer to the OT-2
2m
Import the script we downloaded from (I named it Example-Moclo.py) to the OT-App.
Example-Moclo.py Result of importing the Python script in the OT-App
1m
Run the protocol in the robot that we have transferred the Excel file
,
Labware and liquid set-up layout that corresponds to the run of the example variable input file
Volumes of the different reagents needed to perform the protocol that corresponds to the run of the example variable input file
Positions and volumes of different DNA parts. The volumes are calculated but the positions are the ones set in the input file
1m
Clean the platform of the robot that we are going to perform the protocol
5m
Prepare all reagents and labware in the places as the App is showing, taking into account the notes in step Notes
7m
Start Run
Note
The provided times from this point on are specific to the example, they can variate depending on the ammount of combinations, the variables related to the tip changing, the presence of a thermocycler, etc.
20s
Distributing water
5m 30s
Creation and distribution of mix
7m 30s
Distribution of DNA parts (acceptor and modules)
Expected result
Example of the content of a well, in this case A1 in the labware Final Plate 1 with Combinations Slot 7
Here, we will obtain the modular cloning mix and the diffreent DNA parts that are set in the input file. These positions are seen in the image by the grey wells, and we can see the info of the plate and the media in the plate on slot 7 in this case
1h 28m
Temperature Profile
3h 50m
Retrieve labwares from the OT
5m
Retrieve the final map(s) file from the robot where we run the protocol. In this case, they will be called final_construct_map-example.xlsx (name that is stated in the variable file in the variable Name File Final Constructs located in the GeneralVariablesSheet)
Command line window with the transfer command of the final file with the map(s) from the OT to our computer
final_construct_map-example.xlsx 1m
Protocol references
The Laboratory Automation Protocol (LAP) Format and Repository: A Platform for Enhancing Workflow Efficiency in Synthetic Biology (ACS Synth. Biol.) https://doi.org/10.1021/acssynbio.3c00397
Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria (Nucleic Acids Research) https://doi.org/10.1093/nar/gkad758