Notes
- Standard protocol for preparation of competent cells for transformation with heat-shock method.
- Yields ~200 50uL tubes of cells.
- It is vital to work quickly and keep everything cold and work. Use a cold centrifuge, keep things on ice as much as possible and try to work in a cold room.
- Caution: Uses LN2
Source
- Protocol adapted the Rausher lab
- Used at ACPFG, Adelaide.
Equipment
- Cooling centrifuge.
- Ice machine, or at least cold packs.
- Best results obtained if using a cold room to do final steps, especially pipetting into Eppendorf tubes.
- Supply of liquid N2
Materials
- Antibiotic-free LBA plates – at least 1 per strain + 1 for final competency test.
- Amp/Spec/Kan LBA plates – for test of competency.
- Control plasmid – for test of competency.
- Autoclaved 500 mL flask – for secondary culture.
- Autoclaved 150 ml beaker (with stir bar) – for TS buffer.
- 15 mL Culture/Falcon tubes (sterile) – for overnight culture.
- 50 mL Falcon tube – for resuspention of competent cells.
- Eppendorf tubes (Sterile) – for final competent cells (approx 200)
- Solutions:
- 2x YT Broth (autoclaved)
- TS buffer (TSB) (filter sterilised)
| 2X YT Broth | for 250 mL |
|---|---|
| Bacto tryptone | 4 g |
| Bacto yeast extract | 2.5 g |
| NaCl | 1.25g |
| ddH2O | to 225 mL |
| pH to 7.0 with NaOH | finally, fill to 250 mL with ddH2O |
Method
DAY 1
- Use sterile techniques to streak antibiotic-free LB plate with stock cells (protocol works with DH5-α, TOP-10, DB3.1, etc.).
- Incubate plate at 37°C overnight.
DAY 2
- Autoclave 2X YT broth, 500 ml flask (for secondary culture), 50 ml graduated cylinder (for TS buffer).
- Pick colonies from antibiotic-free LB plates and start an overnight culture in 4mL YT broth in 15 mL falcon tubes. Shake at 37°C overnight.
DAY 3
- Transfer 2 ml of primary culture into 500 ml flask with 200 ml room temperature 2X YT broth (reserve remaining 2X YT broth to use as blank for OD600 reading). Shake at 37°C until OD600 = 0.3-0.6 (2-3 hours).
- Not optimal, but can judge by eye.
- While incubating secondary culture, prepare 15 mL cold TS buffer (TSB).
- Divide secondary culture into four 50 ml tubes. Centrifuge in 20 min at 4°C. Discard supernatant. Keep cells on ice from here on.
- Resuspend cells in 10 ml cold TSB (2.5 ml per tube). Pool cells into a single tube. Incubate on ice 10 min.
- Aliquot into 1.5 ml tubes (50 µl each), on ice. Freeze immediately in liquid N2. Store at -80°C. Warning: Use of LN2 in cold room is dangerous.
- Test for antibiotic resistance by plating tubes of cells (untransformed) on LB/amp and LB/kan plates.
- Use heat-shock method with control plasmid and test for competency.
