Laboratory Protocols
Preparation of competent cells
Making cells competent means preparing the cell wall and cell membrane for the
uptake/throughput of DNA. Either after a heat shock, or by means of electroporation, DNA will
be brought into the cells.
A pre-treatment with CaCl2 or DMSO will lead to a greater efficiency.
Materials
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2 or 3 days in advance, the recipient strain of E. coli is put on minimal medium for E. coli,
and stored at 37 °C)
The night before we make an overnight culture of this so-called “starvation strain” in 2 x
20 ml LB-broth.
For the day of the practical you will need:
- 300 ml 0.1 M Magnesium Chloride (4 °C), sterile
- 100 ml 60% glycerol, sterile
- 250 ml 0.1 M CaCl2 (4 °C), sterile
- 250 ml LB-broth
- Sorvall ss-34 rotor centrifuge
- Sterile Sorvall-tubes
- Shaking water-basin at 37 °C
- Eppendorf centrifuge, Eppendorf tubes
- 0.1 – 1 ml pipettes and tips
- Vortex
Methods
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Keep the cells continuously on melting ice!
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Take your overnight culture of E. coli in LB
Dilute your overnight culture 1/100 in LB (40 ml total for ss-34 tubes, 10 ml for sterile
centrifugation tubes)
Incubate about 2 hours in a shaking water-basin (37 °C) until the Optical Density at 600
nm is 0.3 – 0.5; this means you have about 2 – 4 x 108 bacteria per ml.
Cool the culture on ice and leave for 5 minutes on ice
Spin for 10 minutes at 2500 G in ice-cold sterile tubes (You should be able to calculate
how many rpm this is on the centrifuge you use; there should be a table in your lab where
you can find this). Tare very carefully, tube and holder.
Remove the supernatant; throw it away.
Resuspend the pellet in 0.1 x the volume of ice cold 0.1 mol/l Calcium Chloride solution.
Save for 15 minutes on ice.
Use directly for the transformation; keep it on ice until you need it.
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Laboratory Protocols
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Or store in the –70 °C freezer: put 0.3 ml glycerol per ml cells, ie. a 15% v/v solution,
vortex and make portions of 150 l in Eppendorf tubes. In this way you can store the
competent cells for a long time.