🦠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
Optimized heat shock transformation protocol for Escherichia coli TOP10, JM109, and BL21 strains
🧪 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
📋 Protocol Steps
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.
Add 1 µL of plasmid DNA (10 pg – 100 ng) directly to the cells. Mix gently by tapping or pipetting without vortexing.
Incubate the tube on ice for 30 minutes.
Transfer the tube to a 42 °C water bath for 45 seconds to perform the heat shock.
Immediately return the tube to ice for 2 minutes.
Add 400 µL of SOC medium to the tube.
Incubate at 37 °C for 1 hour with gentle shaking (optional) to allow expression of antibiotic resistance.
Plate an appropriate volume (usually 100 µL) onto LB agar plates with the correct antibiotic, according to the plasmid's resistance marker.
🧾 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.
🧪 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
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.
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.
Harvest cells: Centrifuge the culture at 8000 rpm for 5 minutes at 4 °C.
First resuspension: Discard the supernatant and gently resuspend the pellet in 5 mL of ice-cold 100 mM CaCl₂.
Aliquot: Distribute 1.5 mL of the suspension into three 1.5 mL Eppendorf tubes.
Quick spin: Centrifuge each tube at 8000 rpm for 30 seconds at 4 °C.
Final resuspension: Remove the supernatant and gently resuspend each pellet in 0.5 mL of ice-cold CaCl₂. Flick the tube gently to mix.
(Optional) Add 15% glycerol if preparing for freezing. Store at -80 °C in aliquots.
🧫 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:
First, plate the transformed cells on simple LB agar with the appropriate antibiotic to allow colony growth.
Then, pick a colony and pre-culture it in liquid LB with antibiotic.
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)
Na₂HPO₄
6 g
Buffering agent
KH₂PO₄
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.
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