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Team:Paris Bettencourt/Protocols
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Protocols
Contents |
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.
Protocol 4: E.coli Chromosomal Integration
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Protocol: E. coli Chromosomal Integration (à la Datsenko and Wanner)
- The night before the transformation, start an overnight culture of cells.
5 ml LB Amp.
- The day of the transformation, dilute the cells 100X.
100 ml LB Amp.
Grow at 30°C for about 90 minutes.
- Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8.
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
- Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
- Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
- Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.
Preparation of Electrocompetent Cells
Note: Competent cells should never be vortexted, as this will cause them to lyse
and release salts into the media. Resuspend cells by pipeting up and down with a large
pasteur pipet. Once they are chilled, cells should be continuously cold.
- Float a filter in a Petri dish filled with water.
Millipore membrane filter 0.025 uM.
- Pipet one drop of PCR product onto the filter.
200 ng is needed per transformation.
20 - 100 ul fits well on one filter.
- Collect the drop after 30 - 45 minutes.
The volume will change, but the DNA is not lost.
Dialysis of PCR or Digestion Products
Note: DNA for electroporation must be free of salts to avoid arcing.
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 
