Jan 29, 2024
Salmonella Quick Electroporation Protocol
- 1U.S. Food and Drug Administration, College Park, MD
- Christina M. Ferreira: This work is part of developed methods for the Human Foods Program at the FDA.
Protocol Citation: Christina M. Ferreira, Jie Zheng, Rebeccabell 2024. Salmonella Quick Electroporation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3wxwl47/v1
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
Created: January 11, 2023
Last Modified: January 29, 2024
Protocol Integer ID: 75154
Keywords: Salmonella, electroporation, transformation, electrocompetent
Abstract
Protocol for generating electrocompetent Salmonella cells and transforming them in less than 1-day, with a high transformation efficiency.
Before start
Be sure to have your intended material (plasmid/DNA) to be transformed ready and quantified, as well as all media that you will need for successful completion.
The 10% glycerol used in this protocol should be sterilized by filtration using a .22µm filter, and any water used should be PCR grade sterile water.
Day 1: Preparation of the target strain(s) from -80°C
Day 1: Preparation of the target strain(s) from -80°C
1d
Isolating for single colonies, plate your Salmonella strain onto Tryptic-Soy Agar(TSA).
Incubate at 35°C±2°C for 20-24 hours.
Day 2: Preparation of the Target Strain(s)
Day 2: Preparation of the Target Strain(s)
1d
Inoculate 2ml of Tryptic-Soy Broth (TSB) with a single colony of your strain from your overnight TSA plate.
Incubate at 35°C±2°C, without shaking, for 18-22 hours.
Day 3A: Preparation of Electrocompetent Cells
Day 3A: Preparation of Electrocompetent Cells
3h 30m
In a 125mL Erlenmeyer flask containing 25mL of fresh TSB, transfer 1mL of the overnight culture (1:25 dilution) and incubate at 35°C±2°C, without shaking for 3 hours.
Before the end of the incubation, pre-warm SOC (measured into 1ml microcentrifuge tubes) at 35°C. You will need to make enough 1ml aliquots of SOC for the total number of transformations you plan to do plus 1 for a negative control (1 transformation = 1ml of culture).
Additionally, place sterile water, filter sterilized 10% glycerol and electroporation cuvettes on ice.
Harvest 1mL of your 3-hour culture into microcentrifuge tubes for every transformation you intend to perform plus one additional aliquot for a negative control. Pellet the cells by centrifugation at 13,200rpm for 1 minute.
Discard the supernatant by pouring and then remove any excess with a 200µl pipettor. Immediately place cells on ice.
Resuspend the cell pellet in 1ml of cold sterile water by pipetting gently up and down, making sure not to create any bubbles.
Centrifuge the cell slurry at 13,200rpm for 1 minute.
Discard the supernatant by pouring and then remove any excess with a 200µl pipettor. Immediately place cells on ice.
Resuspend the cell pellet in 1ml of cold, sterile 10% glycerol by pipetting gently up and down, making sure not to create any bubbles.
Centrifuge the cell slurry at 13,200rpm for 1 minute.
Discard the supernatant by pouring and then remove any excess with a 200µl pipettor. Immediately place cells on ice.
Resuspend pellet in 50µl cold sterile 10% glycerol by pipetting gently up and down, making sure not to create any bubbles (50-70µl cell slurry final volume). Immediately place the cell suspension on ice and allow it to remain there for up to 4 hours.
NOTE: Though not advisable, it is possible to freeze the cell suspension at -80°C at this point to use the cells later. The transformation efficiency will not be as robust compared to that of the freshly prepped cells.
Day 3B: Electroporation of Electrocompetent Cells
Day 3B: Electroporation of Electrocompetent Cells
1h 30m
Add the appropriate amount of DNA (Table 1) to your prepped cell suspension using a micropipettor, and mix by gently swirling with your pipet tip. Incubate on ice for 3-5 minutes.
| [DNA]ng | ||
| Plasmid | 100 | |
| dsDNA | 100 |
Table 1: Amont of genetic material to mix with your competent cells.
Transfer the entire cell/DNA slurry to a pre-chilled electroporation cuvette using a P100/200 by pipetting slowly down the side(making sure not to create any bubbles) and then tapping to move cell mixture to bottom.
Electroporate, using the parameters below, for the size cuvette you are using, and immediately add 1ml of pre-warmed SOC to move electroporated cells from the cuvette to the SOC microfuge tube. Incubate at 35°C±2°C (heat block/incubator) for 1 hour, at minimum.
| Cuvette Size | Volts | Resistance | |
| 0.1 cm | 1.8 kV | 200 | |
| 0.2 cm | 2.5 kV | 200 |
NOTE: Please adapt temperature at which you are incubating for any heat sensitive genes or plasmids you are adding or have added into the strain.
NOTE: Recovery time is 1 hour minimum, please adapt for your downstream application in the case of chromosomal insertions which may require more time to complete the recombination.
Without vortexing the recovery but mixing by flicking, spread plate 50µl – 100µl onto LBA or TSA plates containing your selective compound and incubate overnight at 35°C±2°C for 20-24 hours.