Team:Paris Bettencourt/Notebook/Interlab Study

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Paris Bettencourt 2014



Notebook

Interlab Study

July

July 4th

We will participate in the iGEM interlab study: https://2014.igem.org/Tracks/Measurement/Interlab_study

The three specific devices teams are required to measure fluorescence data for are given below.

Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector.
  1. Kit location
    1. Plate 4, Well 18A
E.Coli  NEBTurbo competent cells

To use the DNA in the Distribution Kit you may follow these instructions:

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.
  2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
  3. Transform 1 or 2uL of the resuspended DNA into your desired competent cells,
Heat Shock Transformation of E. coli

This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.

Note: Never vortex competent cells. Mix cells by gentle shaking.
  1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
  2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
    • For an Intact Vector: Add 0.5 ul or less to the chilled cells
  3. Mix gently by flicking the tube.
  4. Chill on ice for 10 minutes. This step is optional, but can improve yields when transforming a ligation product.
  5. Heat shock at 42 °C for 30 seconds.
  6. Return to ice for 2 minutes.
  7. Add 200 ul LB medium and recover the cells by shaking at 37 °C.Kanamycin: 120 minutes
  8. Plate out the cells on selective LB.Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
  9. Incubate at 37 °C. Transformants should appear within 12 hrs.
Result: Bacteria grow and made big ans small colonies

     4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.







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August

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September

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October

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