TITLE: Salmonella Quick Electroporation Protocol
AUTHORS: Christina M. Ferreira, Jie Zheng, Rebeccabell

[DESCRIPTION]
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.  



[STEPS]
SECTION: Day 3A: Preparation of Electrocompetent Cells
5. 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.
SECTION: Day 3A: Preparation of Electrocompetent Cells
6. 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.
SECTION: Day 3A: Preparation of Electrocompetent Cells
7. 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.

SECTION: Day 3A: Preparation of Electrocompetent Cells
9. Resuspend the cell pellet in 1ml of cold sterile water by pipetting gently up and down, making sure not to create any bubbles.
SECTION: Day 3A: Preparation of Electrocompetent Cells
12. Resuspend the cell pellet in 1ml of cold, sterile 10% glycerol by pipetting gently up and down, making sure not to create any bubbles.
SECTION: Day 3A: Preparation of Electrocompetent Cells
10. Centrifuge the cell slurry at 13,200rpm for 1 minute.
SECTION: Day 3A: Preparation of Electrocompetent Cells
15. 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.
SECTION: Day 3B: Electroporation of Electrocompetent Cells
16. 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.

            
 

SECTION: Day 3B: Electroporation of Electrocompetent Cells
17. 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.
SECTION: Day 3B: Electroporation of Electrocompetent Cells
18. 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.

 

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.

SECTION: Day 3B: Electroporation of Electrocompetent Cells
19. 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.
SECTION: Day 2: Preparation of the Target Strain(s)
3. Inoculate 2ml of Tryptic-Soy Broth (TSB) with a single colony of your strain from your overnight TSA plate.
SECTION: Day 1: Preparation of the target strain(s) from -80°C
2. Incubate at 35°C±2°C for 20-24 hours.  
SECTION: Day 2: Preparation of the Target Strain(s)
4. Incubate at 35°C±2°C, without shaking, for 18-22 hours. 
SECTION: Day 1: Preparation of the target strain(s) from -80°C
1. Isolating for single colonies, plate your Salmonella strain onto Tryptic-Soy Agar(TSA). 
SECTION: Day 3A: Preparation of Electrocompetent Cells
8. Discard the supernatant by pouring and then remove any excess with a 200µl pipettor.  Immediately place cells on ice.
SECTION: Day 3A: Preparation of Electrocompetent Cells
11. Discard the supernatant by pouring and then remove any excess with a 200µl pipettor.  Immediately place cells on ice.
SECTION: Day 3A: Preparation of Electrocompetent Cells
14. Discard the supernatant by pouring and then remove any excess with a 200µl pipettor.  Immediately place cells on ice.
SECTION: Day 3A: Preparation of Electrocompetent Cells
13. Centrifuge the cell slurry at 13,200rpm for 1 minute.