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From 2014.igem.org

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Notebook
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Lab Protocols

Competent Cell with Calcium Chloride

1. Materials

• Solid LB medium - 1 plate
• Liquid LB medium
• 2 250ml centrifuge tubes
• Centrifuge
• Cell sample (DH5-alpha in our case)
• Autoclave
• Inoculation loop
• Liquid nitrogen
• 37°C oven
• CaCl₂:
• 60mM CaCl₂
• 10mM HEPES
• 15% glicerol
• H₂O to 100ml

2. Method

Day 1

1. Risk the LB plate with the cell sample (DH5-alpha)
2. Incubate the plate at 37°C overnight

Day 2

1. Sterilize in the Autoclave
• 100ml of liquid LB medium
• 100 ml of CaCl₂
• 2 250ml centrifuge tubes
2. Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight

Day 3

1. Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375
2. Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C
3. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution
4. Spin the tubes at 7000rpm/7min/4°C
5. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution. Incubate 30min on ice
6. Spin at 7000rpm/7min/4°C
7. Discard the supernatant and ressuspend the pellet in 2ml of the CaCl₂ solution
8. Distribute the 2ml in 500ul tubes adding 50ul in each.
9. Freeze the samples in liquid nitrogen
10. Stock at -80°C