Team:Brasil-SP/Notebook

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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. Methods

    3. 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

Transformation in Escherichia coli (DH5-alpha)

  1. Materials

    • 1,5ml tube
    • Styrofoam box with ice
    • Agar plate with antibiotic
    • Competent cells
    • Plasmidial DNA
    • Centrifuge
    • Water bath at 42°C
    • Liquid LB medium
    • Shaker

  2. Methods

    1. Briefly spin the competent cells and put then on ice
    2. Add 50ng of plasmidial DNA in a 1,5ml tube
    3. Add the 50ul of competent cell in the same tube
    4. Keep the tube on ice for 25min
    5. Put the tube in a 42° water bath for 2 min
    6. Put the tube on ice for 5 min
    7. Add 200ul of liquid LB
    8. Incubate at 250rpm/37°C/1 hour
    9. Plate the the solution in a Agar plate with the appropriate antibiotic
    10. Incubate the plate at 37°C overnight

Assemblies

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