This post introduces a simple, economical, and surprisingly effective protocol for preparing TSS competent cells—chemically competent E. coli prepared using the TSS (Transformation and Storage Solution) method. Originally described by Chung et al. (1989), the TSS competent cell preparation method remains a low-cost, high-utility standard in molecular biology labs.
Why TSS Competent Cell Preparation Still Matters
- All-in-One Protocol: TSS supports both preparation and storage in a single solution
- Cost-Effective: No need for electroporation or expensive kits
- Reliable Efficiency: ~10⁶ CFU/µg DNA (fresh)
- Ideal for Cloning: Works well for routine plasmid transformation
TSS competent cell preparation is ideal for labs that need quick, affordable, and moderately efficient transformation protocols—especially for E. coli.
Materials: TSS Solution Recipe
- 85% LB medium
- 10% PEG 8000 (w/v)
- 5% DMSO (v/v)
- 50 mM MgCl₂ (adjust pH to 6.5)
- Sterilize by autoclaving or filtering
- Store at 4°C for up to 2 weeks
🔬 Step 1: Preparing TSS Competent E. coli
- Streak E. coli on LB agar plate (add antibiotic if needed) and incubate at 37°C overnight.
- Inoculate a well-isolated colony into 5 ml LB broth (with antibiotic) and shake at 37°C overnight at 220 rpm.
- Dilute 1 ml of this culture into 100 ml fresh LB (no antibiotic) in a sterile 500 ml flask.
- Incubate at 37°C with shaking until OD₆₀₀ ≈ 0.5 (2–2.5 hr).
- Chill the culture on ice for 20 minutes.
- Harvest by centrifuging at 1500 rpm, 5 minutes, 4°C.
- Resuspend the pellet in 10 ml ice-cold TSS solution.
You now have fresh TSS competent cells. Proceed directly to transformation or store as follows:
- Short-term: Keep on ice or at 4°C (up to 6 hours)
- Long-term: Flash-freeze in liquid nitrogen or dry ice, store at −70°C
⚠️ Frozen cells retain ~30% of transformation efficiency compared to fresh TSS competent cells.
Step 2: Chemical Transformation with TSS Competent Cells
- Add plasmid DNA (≤20 µl) to 150 µl of ice-cold TSS competent E. coli.
- Incubate on ice for 30 minutes, mixing gently a few times.
- Heat shock for 2 minutes at 42°C.
- Return to ice for 2 minutes.
- Add 800 µl LB broth (or SOC for better recovery).
- Incubate at 37°C for 60 minutes with shaking.
- Plate ≤200 µl onto LB agar with appropriate antibiotic.
- Incubate overnight at 37°C.
✅ For higher efficiency, SOC medium can be used:
SOC Recipe: 2% Bactotryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose.
📊 Fresh vs Frozen TSS Competent Cells
| Storage Condition | Efficiency (CFU/µg DNA) | Notes |
|---|---|---|
| Fresh (4°C ≤ 6 hr) | ≥ 1×10⁶ | Highest transformation rate |
| Frozen (−70°C ≤ 2 mo) | ~3×10⁵ | Use snap-freeze to preserve |
📚 Reference & Further Reading
- DOI: https://doi.org/10.1073/pnas.86.7.2172
- Chung, C. T., Niemela, S. L., & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. PNAS, 86(7), 2172–2175. https://doi.org/10.1073/pnas.86.7.2172
- Addgene Protocol Repository: https://www.addgene.org/protocols/
🧠 Final Thoughts
TSS competent cell preparation may not have the ultra-high efficiency of commercial kits or electroporation, but its balance of simplicity, affordability, and decent transformation efficiency keeps it relevant today. Whether you’re running a budget-tight lab or teaching undergraduates, this protocol remains a time-tested tool.
💬 Discussion Starters
Q1. Have you optimized TSS for different E. coli strains such as BL21 or TOP10?
Q2. What transformation efficiency do you typically achieve with TSS-prepared cells?
Q3. Could this method be adapted for non-E. coli species with membrane modifications?
