Preparation of chemically competent cells. This protocol involves steps occurring over multiple days — prepare accordingly.
Materials
- TSS buffer (recipe below)
- LB broth
- Bacterial stock (e.g. DH5-α)
- 1.5 mL microcentrifuge tubes
Day 1
- Streak bacteria from a glycerol stock on an LB agar plate and incubate at 37°C overnight.
Day 2
- Inoculate a 5 mL LB broth culture, with no antibiotic, using a single colony from the plate in step 1. Incubate the culture overnight at 37°C.
- Prepare TSS buffer using the following recipe:
| Reagent |
Amount |
| PEG 8000 (or 3350) |
5 g |
| MgCl₂, 1 M ¹ |
1.5 mL |
| DMSO |
2.5 mL |
| LB broth |
up to 50 mL |
¹ 0.3 g MgCl₂·H₂O can be used in place of the 1 M MgCl₂ solution.
- Filter the TSS buffer through a 0.22 μm filter and store at 4°C.
Day 3
- Dilute the culture 1:100 by inoculating four 50 mL conical tubes, each with 50 mL LB broth and 500 μL of overnight bacterial culture. Grow the cultures at 37°C until the OD₆₀₀ is between 0.2 and 0.5.
- Chill the culture, 1.5 mL microcentrifuge tubes, and TSS on ice for 10 minutes. Meanwhile, cool the centrifuge to 4°C.
- Pellet the bacterial cells at >3000 RCF for 10 minutes at 4°C.
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The cells must remain on ice and stay cold from this point forward in order to maintain a high transformation efficiency.
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- Remove the supernatant and resuspend in 10% volume chilled TSS buffer — for example, resuspend a 50 mL culture in 5 mL TSS buffer.