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 make Competent E. coli 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
How to Make TSS Competent Cells: Step-by-Step Protocol for Reliable Transformation
🔬 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.
✅ FAQ 섹션
Q. How do I make TSS competent E. coli cells in the lab?
A. To make TSS competent cells, begin by culturing E. coli to mid-log phase (OD600 ~0.5), then chill and resuspend the cells in ice-cold TSS buffer containing LB, PEG 8000, DMSO, and MgCl₂. This buffer enables both transformation and storage in one step, making it ideal for routine cloning tasks.
📊 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 |
While fresh TSS competent cells offer the highest transformation efficiency—often exceeding 1×10⁶ CFU per microgram of DNA—they are limited by a short usable window. In contrast, frozen cells provide greater convenience and flexibility for scheduling but come with a significant drop in efficiency, typically retaining only ~30% of the activity. Proper snap-freezing and storage at −70°C are essential to preserving functionality for up to two months without substantial degradation.
📚 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: Why This Still Matters
This protocol is based on my own experimental lab notes from undergraduate and early graduate research, refined through repeated use in a real-world, resource-limited setting. I’ve personally used the TSS method to prepare competent E. coli across dozens of cloning projects, and despite its simplicity, it consistently delivered reliable transformation rates, especially for routine plasmid work where speed and cost-efficiency are paramount.
In an era of high-throughput commercial kits and automated systems, it’s easy to overlook foundational methods like TSS competent cell preparation. But their value remains undeniable: they teach hands-on biochemical reasoning, demand attention to sterile technique, and democratize access to core molecular tools without depending on expensive infrastructure.
Whether you’re a student learning transformation for the first time, a DIY biologist, or a cost-conscious researcher, this protocol stands as a testament to how thoughtful, low-cost methods can still compete—if not outperform—in specific contexts.
