IGEM:VGEM/2007/Notebook/2007-8-7

• Transforming DB3.1 Cells:

1. Thaw competent cells on ice. Place required number of 17 × 100 mm polypropylene tubes on wet ice.
2. Gently mix cells, then aliquot 100 µl competent cells into chilled polypropylene tubes.
3. Refreeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning them to the -80°C freezer. Do not use liquid nitrogen.
4. Add 5uL of biobrick construction plasmid DNA to cells, moving pipet through cells to mix. Gently tap to mix.
5. Incubate cells on ice for 30 minutes.
6. Heat-shock cells 45 seconds in a 42°C water bath; do not shake.
7. Place on ice for 2 minutes.
8. Add 0.9 ml of room temperature S.O.C. Medium (Cat. No. 15544-034).
9. Shake at 225 rpm (37°C) for 1 hour.
10. Plate transformed cells @ 75uL and 150 uL on ampicillin plates
11. Plate control cells @ 100uL on control and ampicillin plates (for a total of 8 plates)
12. Incubate overnight at 37°C.

• Butanol Tolerance
  • Plated 1.6% butanol cells.
  • Added yesterday's 1.6% butanol tolerant cells to 3.2% butanol broth solution.
  • Put 3.2% solution cells in shaker at 37oC overnight