iGEM Protocol for 2007
- Day 1: Transformation protocol (6/12/07)
- Thaw the 6 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended.
- OMPA1 in PET
- OMPA1 + his in PET
- OMPA1 + strep2 in PET
- OMPA2 in PET
- OMPA2 + his in PET
- OMPA2 + strep in PET
- Add 1 µl of the DNA solution directly to the cells. Stir gently to mix.
- Place the tubes on ice for a little more than 5 minutes
- Heat the tubes for about 45 seconds in a 42°C water bath; do not shake
- Place on ice for 2 minutes
- Add 250 µl of room temperature SOC Medium to each tube
- Shake at 37°C (300 rpm) for more than 30 minutes.
- Light the bunsen burner
- Using two sets of petri dishes containing LB agar w/ Kanamycin (1 w/ 5 µl of cells and one w/ 50 µl of cells)
- 5 µl: pipette 5 µl of cells with 45 µl of SOC onto the agar mix
- 50 µl: pipette 50 µl of cells onto the agar mix.
- For each plate, we use sterile technique to spread the agar:
- Take the metal spreader and soak the bottom in ethanol, making sure to rinse exposed parts of the handle with distilled water.
- Very briefly put the ethanol soaked parts of the spreader under the flame.
- Touch the agar to make sure the spreader isn't too hot (will singe if too hot), and spread the cells around the plate.
- Afterwards, turn the plates upside down and incubate at 37°C
Protocol Recorded by Kevin Shee