> For the complete documentation index, see [llms.txt](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/bacterial-transformation.md).
# BACTERIAL TRANSFORMATION
This protocol describes the chemical transformation using the classic heat shock method. It is suitable for introducing plasmid DNA into competent cells prepared
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[Optimized heat shock transformation protocol for Escherichia coli TOP10, JM109, and BL21 strains](https://dx.doi.org/10.17504/protocols.io.yxmvmm6wbv3p/v1)
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### 🧪 Materials
* Chemically competent bacteria (50 µL per reaction)
* Plasmid DNA (10 pg – 100 ng in ≤1 µL volume)
* SOC medium (or LB if SOC is not available)
* Sterile microcentrifuge tubes (1.5 mL)
* Water bath or heat block set to 42 °C
* Ice bucket
* Incubator at 37 °C
* Agar plates with the appropriate antibiotic
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### 📋 Protocol Steps
1. **Thaw competent cells** on ice. Add **50 µL** of competent *E. coli* to a pre-chilled 1.5 mL tube and keep on ice for **5–10 minutes**.
2. Add **1 µL of plasmid DNA** (10 pg – 100 ng) directly to the cells. Mix **gently** by tapping or pipetting **without vortexing**.
3. Incubate the tube on ice for **30 minutes**.
4. Transfer the tube to a **42 °C water bath** for **45 seconds** to perform the heat shock.
5. Immediately return the tube to **ice for 2 minutes**.
6. Add **400 µL of SOC medium** to the tube.
7. Incubate at **37 °C for 1 hour** with gentle shaking (optional) to allow expression of antibiotic resistance.
8. Plate an appropriate volume (usually 100 µL) onto **LB agar plates with the correct antibiotic**, according to the plasmid's resistance marker.
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### 🧾 Notes
* The exact amount of DNA depends on the plasmid size and concentration. Avoid adding more than 1 µL of DNA to preserve transformation efficiency.
* SOC medium promotes higher transformation efficiency but can be replaced with LB if necessary.
* Incubation after transformation is essential for expression of antibiotic resistance genes before plating.
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### 🧪 Preparation of Chemically Competent *E. coli* Cells (CaCl₂ Method)
This protocol describes the preparation of chemically competent *E. coli* cells using calcium chloride, suitable for transformation via heat shock. It is a quick and cost-effective method for routine lab use.
**Protocol Steps**
1. **Overnight culture**: Inoculate the desired *E. coli* strain in **0.5 mL LB medium** and grow overnight in **50 mL LB** in a Greiner or similar tube at 37 °C with shaking.
2. **Inoculation**: Transfer **0.1 mL of the overnight culture** into **10 mL fresh LB** and grow at 37 °C until the culture reaches **OD₆₀₀ ≈ 0.6**.
3. **Harvest cells**: Centrifuge the culture at **8000 rpm for 5 minutes** at 4 °C.
4. **First resuspension**: Discard the supernatant and gently resuspend the pellet in **5 mL of ice-cold 100 mM CaCl₂**.
5. **Aliquot**: Distribute **1.5 mL** of the suspension into **three 1.5 mL Eppendorf tubes**.
6. **Quick spin**: Centrifuge each tube at **8000 rpm for 30 seconds** at 4 °C.
7. **Final resuspension**: Remove the supernatant and gently resuspend each pellet in **0.5 mL of ice-cold CaCl₂**. Flick the tube gently to mix.
8. (Optional) Add **15% glycerol** if preparing for freezing. Store at -80 °C in aliquots.
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### 🧫 Autoinduction Medium (Optional – for Protein Expression on Plates)
If the transformation is first performed in **maintenance strains** (such as *E. coli* TOP10 or JM109), it is necessary to carry out a **secondary transformation** into an **expression strain** (e.g., BL21(DE3)) before using the autoinduction medium for protein production.
Alternatively, if the plasmid is directly transformed into an **expression strain**, we recommend the following workflow:
1. First, plate the transformed cells on **simple LB agar** with the appropriate antibiotic to allow colony growth.
2. Then, pick a colony and **pre-culture it in liquid LB** with antibiotic.
3. Finally, **subculture onto autoinduction plates** for protein expression.
This ensures healthy initial growth and optimal protein expression conditions.
🧪[ **Composition of 4× YT Autoinduction Medium (1 L final volume)**](https://www.protocols.io/view/plate-protein-expression-on-autoinduction-media-q26g78n91lwz/v3?step=13.1)
| Naâ‚‚HPOâ‚„ | 6 g | Buffering agent |
| ----------------- | ------------------- | ------------------------------------------------- |
| KHâ‚‚POâ‚„ | 3 g | Buffering agent |
| Tryptone | 20 g | Nitrogen source |
| Yeast Extract | 5 g | Growth factors and vitamins |
| NaCl | 5 g | Ionic balance |
| Agar | 15 g | Solidification agent |
| **50% Glycerol** | 10 mL | Carbon source |
| **10% Glucose** | 5 mL | Initial carbon source (represses early induction) |
| **5% Lactose** | 20 mL | Inducer for protein expression |
| **Sterile Water** | To 1 L total volume | Diluent |
After autoclaving the base medium (containing agar and salts), add the sterile sugar solutions (glycerol, glucose, lactose) and the corresponding antibiotic (**e.g., Kanamycin 50 µg/mL**) when cooled to approximately 50 °C. Pour into Petri dishes and let solidify.
This medium facilitates **autoinduced protein expression**, eliminating the need for IPTG by leveraging the metabolic switch from glucose to lactose.