E. coli Competent Cell Preparation and Transformation
Núria Crua· Postdoc· University of Lausanne (UNIL)
Published Jul 27, 2026~1h 59m total· 1 contributions
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JNAR-ID: JNAR-69755530
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PublishedLast updated Jul 27, 2026
Objective
Preparation of chemically competent E. coli cells using calcium chloride and their subsequent transformation with plasmid DNA.
Classification
Sample typeDNA
TechniqueCloningTransfection
Experimental contextIn vitro
Biological modelBacteria
Reproducibility status
Reproducible
No reproducibility blockers or risks reported. The protocol runs end-to-end.
20 healthy
⏱ Total 1 h 59 min3 sections20 steps
Day before
1
Add a colony of E.coli, or material from a glycerol stock, to 10 ml of LB (Luria-Bertani) medium.
2
Incubate overnight with shaking 250 rpm at 37 °C.
Day of the protocol — Competent cell preparation
25 min
3
Make an E.coli fresh culture ( 1:100 diluting the ON, 200 ul of culture in 20 mL of LB) and incubate it at 37 ºC and 250 rpm shaking until it reaches OD 600nm = 0.6-0.7
Critical stepThe culture must be harvested at the correct OD (0.6-0.7) for optimal competency.
4
Pre-cool the centrifuge and all the material to be used.
5
Centrifuge the cultures for 10 minutes at 4000 rpm (8000 g) at 4 °C.10 min
6
Discard the supernatant and add 5 ml of autoclaved 0.1 M CaCl2 to the pellet (bacteria).
7
Keep the bacteria on ice for 10 minutes.10 min
8
Add 5 ml of autoclaved 0.1 M CaCl2 to the pellet and centrifuge 5 minutes at 2500 rpm at 4 °C.5 min
9
Discard the supernatant and add 1 ml of cold 0.1 M CaCl2 to the pellet.
10
Aliquot the cells into pre-cooled Eppendorf tubes at 50 µl each, label them, and store at -80 °C.
Transformation
1 h 34 min
11
Prepare the material: water bath at 42 °C, ice, warm the LB, and pre-cool the material.
12
Add about 5 µl of plasmid at an approximate concentration of 50-100 ng/µl.
13
Place on ice for 30 minutes, tapping with a finger every 5/10 minutes to mix well.30 min
14
Place the Eppendorf tube at 42 °C for 50 seconds (heat shock).
Critical stepThe heat shock timing (50 seconds at 42 °C) must be precise for efficient transformation.
15
Quickly remove the Eppendorf tube from the bath and leave it on ice for 2 minutes.2 min
Critical stepTransfer must be done rapidly and the 2 minutes on ice observed exactly.
16
After exactly two minutes, add 1 ml of LB (previously warmed).
17
Grow the cultures at 37 °C with shaking for 1 hour.1 h
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