- Transforming DB3.1 Cells:
- Thaw competent cells on ice. Place required number of 17 × 100 mm polypropylene tubes on wet ice.
- Gently mix cells, then aliquot 100 µl competent cells into chilled polypropylene tubes.
- 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.
- Add 5uL of biobrick construction plasmid DNA to cells, moving pipet through cells to mix. Gently tap to mix.
- Incubate cells on ice for 30 minutes.
- Heat-shock cells 45 seconds in a 42°C water bath; do not shake.
- Place on ice for 2 minutes.
- Add 0.9 ml of room temperature S.O.C. Medium (Cat. No. 15544-034).
- Shake at 225 rpm (37°C) for 1 hour.
- Plate transformed cells @ 75uL and 150 uL on ampicillin plates
- Plate control cells @ 100uL on control and ampicillin plates (for a total of 8 plates)
- 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