IGEM:VGEM/2007/Notebook/2007-6-19

June 19, 2007

• George- paper presentation
• Emre - paper presentation
• Amy and Kevin: Selecting biobrick for today's transformation (BBa_I13521, Ptet mRFP. switch off by tetracycline)
• Emre, George, Ranjan: Preparing for meeting tomorrow with Morgan Estabrook
• Transformation Protocol of E.Coli with RFP

Getting the DNA out of the Biobrick wells:

  1. Puncture a hole through the foil with a pipette tip into the well that corresponds to Part BBA I13521: Ptet mRFP. switch off by tetracycline.
  2. Add 15 uL of diH2O (deionized water) to the well

Transforming Cells:

  1. Thaw ~50 μl cells on ice. Do not use glass tubes, which adsorb DNA.
        1. Thaw another 50 μl of cells for the control plates (these will not be transformed)
  2. Add 1 ul DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
  3. Incubate all cells on ice for 30 minutes.
        1. Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 50 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 1mL of LB broth to a tube and add all of the cells into it
  7. Incubate for 1 hour at 37°C on shaker. 
        1. Note: Can also save some time here by reducing incubation to ~45 min.
        2. Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
  8. Sterilize the spreader
  9. Spread 20 & 200 μl of transformed cells and control cells onto plates made with appropriate antibiotic (i.e. 100mg/ml of Amp), four plates in total.
 10. Grow overnight at 37°C.