Notebook:Federico Castro M/2008/01/12

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Proposed Procedure

  • Status: Pending for aproval
  • Issues: None

I want to recover 2 biobricks from the kit plates in order assemble the Repressilator. This will improve my skills and the knowledge of how oscillators work and the individual parts might be latter usefull.

The whole procedure should be easy cheap and educative. I await for approval for this procedure.

Biobrick to be extracted

  • BBa_I5612 available at well 6L at iGEM 2007 Parts Kit Plate 1 in plasmid pSB4A3.
  • BBa_I6032 available at well 23K at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1A2.
Repressilator No2

The following protocol is proposed for biobrick recovery from the iGEM page [1]

  1. Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick™-standard part that you want
  2. Add 15 uL of diH2O (deionized water)
  3. Take 1uL DNA and transform into your desired competent cells, plate out on a plate with the correct antibiotic* and grow overnight. Your goal here is to obtain single colonies.

I intend to use the following protocol for transforming the biobricks into competent cells.

  1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
  2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
  3. Incubate on ice for 30 minutes
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 4 volumes of room temperature SOC (not critical)
  7. Incubate for 1 hour at 37°C on shaker.
    • Note: Can also save some time here by reducing incubation to ~45 min.
    • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
  8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37°C.

Before the procedure I would need to prepare solid plates with Ampicillin (The vector of the biobrick has a region that confers the bacteria with Ampicilin resistance). It seems that the proper concentration of Ampicillin is 50 μg/mL.[2]