IGEM:Harvard/2007/Protocols

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iGEM Protocol for 2007

• Day 1: Transformation protocol (6/11/07)

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

• Day 2: Growing Bacteria in Liquid Medium (6/12/07)

1. Take out the 6 grown colonies.
2. Get 6 100 mL culture tubes.
3. In each tube, use the pipet gun to add 2 mL of KB, and add 2 µl of Kanamycin (50 mg/mL)1000x
4. Light the bunsen burner.
5. Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
6. Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
7. Repeat this sterile technique with the other 6.
8. Shake at 37°C (300 rpm) for more than 30 minutes.
9. Incubate overnight

Protocol Recorded by Kevin Shee