Yeast Transformation Protocol

To carry out this high-throughput protocol, you will need the following:

Reagents (Volumes for one sample)	Opentrons Equipment
1 M Lithium acetate (LiOAc)(18 µl)	2x 96-well plates
10 mg/ml Salmon sperm DNA (SSD)(5 µl)	2x Temperature modules (GEN 2)
50% PEG-3350 (120 µl)	4x 24 Eppendorf rack
DNA mini-prep	12-well Reservoir
Yeast cells at appropriate OD (32 µl)	p300 Multi pipette (GEN 2)
5 mM Calcium chloride (CaCl2) (200 µl)	2x 300 µl Tiprack
	p20 Single pipette (GEN 2)
	20 µl Tiprack

Download the folder from GitHub

Creating your customised protocol

Open terminal or command line and change the directory (‘cd’):

$ cd YourFilePath/Yeast_Transformation_OT-main/Opentrons_scripts 

Run the script using python by typing the following for MacOS:

$ python YeastTransformationProtocol_API2.py

Or Windows:

$ python YeastTransformationProtocol_API2

Prompts will appear, as shown below. Enter the specific values for your experiment.

Ignore the following errors (They appear because the computer is not connected to an Opentrons):

$ /.opentrons/deck_calibration.json not found. Loading defaults
$ /.opentrons/robot_settings.json not found. Loading defaults

Enter new file name.

The new file will be saved in the Opentrons_scripts folder.

You can now run this in the Opentrons app!

Creating a separate file for your experiment helps to protect the original script from user-specific changes.

Opentrons set up

Please set up your Opentrons as shown in the image above. Place a 96 well plate onto the cold block.

The reservoir wells should contain:

Reservoir Well	Reagent	Volume
A2	LiOAc	1.8 ml
A2	SSD	475 µl
A4	50% PEG-3350	11.5 ml
A6	CaCl2	19.5 ml
A8	Yeast cells	3.2 ml
A9	Water	20 ml
A12	Waste

You can adjust these volumes for the number of samples you are transforming.

Automated protocol

1. Adds LiOAc (18 µl) and SSD (5 µl) to all wells of 96 well plate on cold block
2. 120 µl PEG is added to all wells
3. DNA mini-preps will be added to specific wells
4. 32 µl yeast cells are added and mixed thoroughly
5. User must move 96 well plate onto hot block
6. Cells undergo heat shock
7. Opentron transfers cells to clean 96 well plate
8. User centrifuges plate and returns to deck
9. Supernatant is removed
10. Cells are mixed with 200 µl CaCl₂
11. Plate can be removed for incubation and plating

Further help

Click the image to watch a detailed demonstration of how to download and implement this code.

Contributors

Bethan Highley,Eloise O'Connor,Jemima Onime,Kimberley A.Owen,Daniella Pretorius,Emily Tsang