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Team:Paris Bettencourt/Protocols
From 2013.igem.org
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Protocols
Protocol 1: Heat Shock Transformation
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Thaw 20 ul of Chemically Competent Cells on Ice
Add 2 ul DNA (intact plasmid)
Incubate on Ice for 30 minutes
Incubate at 42C for 45 seconds
Incubate on Ice for 2 minutes
Add 200 ul of LB broth
Incubate at 37C for 1 hour in shaker
Plate on Agar supplemented with appropriate antibiotics.
Protocol 2: CaCl2 Competent Cells
This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.
Note: Never vortex competent cells.
Resuspend by pipetting with large Pasteur pipettes.
- The night before, inoculate a 5 ml culture and grow overnight with selection.
The day of:
- Dilute cells ~ 1:200 into selective media.
For this example add 250 ul to 50 ml of selective media.
Note: The protocol is easily scaled to increase the number of cells. - Grow the cells to an OD600 of 0.6 – 0.7.
Use a large flask, 500ml, for good aeration.
Use a baffled flask for fastest growth.
This takes about 3 hours depending on the cells.
Medium-heavy cloudiness by eye is fine. - Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
- Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
- Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
- Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
- Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
The night before:
Protocol 3: Glycerol Stocks
- Pick Single colonies from agar plates
- Innoculate 5ml LB broth overnight.
- Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
- Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
+33 1 44 41 25 22/25
team2013@igem-paris.org 
