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CaCl₂ Competent Cell Preparation (50 mL Culture)
Materials
Autoclaved LB broth
Sterile 100 mM CaCl₂ (ice-cold; Use fresh CaCl₂ and autoclaved water for solutions).
Sterile 100% glycerol (if storing)
Sterile 50 mL Falcon tubes or centrifuge tubes
Ice, refrigerated centrifuge
E. coli strain (glycerol stock or plate culture)
🧪 Protocol
1. Inoculation and Growth
Inoculate 3 mL LB with your E. coli strain and grow overnight at 37°C, 200 rpm.
Next day, add 0.5 ml into 50 mL fresh LB in a 250 mL flask.
Grow at 37°C, 200 rpm until OD₆₀₀ ≈ 0.4–0.6 (mid-log phase, ~2–3 hours depending on strain).
2. Chill and Harvest
Transfer the culture to a pre-chilled 50 mL Falcon tube (Keep everything ice-cold after log-phase growth).
Place on ice for 10–15 min to cool the cells (this step increases competency).
Centrifuge at 4,000 rpm (~3,000 × g), 10 min, 4°C.
3. Wash with Ice-Cold CaCl₂
Carefully discard the supernatant.
Gently resuspend the pellet in 25 mL ice-cold 100 mM CaCl₂ (Handle gently; avoid vortexing or harsh pipetting).
Keep on ice for 10–20 min.
Spin again at 4,000 rpm, 10 min, 4°C.
4. Final Resuspension
Discard supernatant gently.
Add Glycerol to a final percentage of 15% to 2 mL ice-cold 100 mM CaCl₂.
Aliquot 100 µL into pre-chilled microfuge tubes.
Use immediately for transformation or snap-freeze in liquid N₂ and store at −80°C.
2. Transformation Protocol: CaCl₂-Competent E. coli
Materials
CaCl₂-competent E. coli (fresh or stored at −80°C)
Plasmid DNA (1–10 ng is enough)
LB broth
Antibiotic-containing LB agar plates
1.5 mL microfuge tubes
42°C water bath
Ice
🧪 Step-by-Step Protocol
1. Thaw Competent Cells
Take 100 µL aliquot of competent cells on ice.
Let thaw for 5–10 minutes.
2. Add Plasmid DNA
Add 1–2 µL of plasmid DNA (do not exceed 10% of the total volume).
Gently flick or tap the tube to mix.
Do not vortex.
3. Incubate on Ice
Incubate the mixture on ice for 30 minutes.
This allows DNA to bind to the cell surface.
4. Heat Shock
Place tubes in a 42°C water bath for 45–60 seconds.
Do not exceed 60 seconds.
Immediately return to ice for 2–5 minutes.
5. Recovery
Add 450–950 µL SOC or LB medium (pre-warmed to room temperature is fine).
Incubate at 37°C for 45–60 minutes with shaking (200–250 rpm).
This allows cells to express the antibiotic resistance gene.
6. Plating
Plate 50–200 µL on LB agar plates with the appropriate antibiotic.
For higher efficiency, spin down the cells, remove most supernatant, resuspend in ~100 µL, and plate the entire amount.
Incubate plates overnight at 37°C.
> Include a no-DNA control to check for antibiotic selection accuracy.
> Use fresh, well-dried plates to avoid spreading.
> For low-copy plasmids or difficult constructs, extend the recovery to 1.5 hrs.
3. Small scale protein expression check
Preparation of pre-inoculum:
Inoculate a single colony in 3mL of LB media with appropriate antibiotics.
Incubate at 37oC overnight.
Small Scale expression check:
Inoculate 3 ml LB media in 50 ml falcon tube with 1% of pre-inoculum and appropriate antibiotics for induced and uninduced expression (Control) and incubate at 37oC for 3 hrs.
Expression was induced in one sample with 1 mM IPTG and incubate both un induced and induced at 37oC for 3 hrs. The culture was centrifuged at 4000 rpm for 10 minutes and the supernatant was discarded.
The pellet corresponding to 3 ml was resuspend in 0.5 ml of lysis buffer followed by sonication for 2 minutes with 3 sec ON and 5 sec OFF.
After sonication, the lysate was centrifuged at 13000 rpm for 15 minutes. The supernatant was transferred to a fresh tube.
The pellet is resuspended in 100 ul of buffer with 2X loading dye.
5/10 ul and 20/ 40 ul of clarified supernatant of induced and un-induced samples were loaded onto the SDS-PAGE.
Additionally, once the strain is shortlisted, Expression can be determined at other temperatures (Eg 18°C), post IPTG addition, 50 ml falcon tubes containing 3 ml of culture needs to be incubated at the corresponding temperatures (Eg 18°C) overnight.
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