IGEM:Harvard/2007/Kevin's Notebook

Growing Bacteria in Liquid Medium (6/27/07)

1. Take out colonies:
   1. OMPA1+his
   2. OMPA2+his
   3. OMPA1+strep
   4. OMPA2+strep
2. Get 4 100 mL culture tubes.
3. Light the bunsen burner.
4. In each tube, use the pipet gun to add 6 mL of KB, and add 6 µl of Kanamycin (50 mg/mL)1000x
   1. Make sure to lightly graze the tip of the KB with the top unscrewed a little for additional sterilization
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) overnight

Innoculation and Induction (6/28/07)

1. Take out colonies from overnight incubation, as well as competent cell line BL21DE3.
   1. Two new culture tubes for each colony, and one for the competent cells.
2. Add 5 ul of Kanamycin (in all but competent cells)
3. Add 5 ml of LB (3 ml for competent cells)
4. Add 200 ul of bacteria (50 ul of competent cells)

1. Add 5 ul of IPTG
   1. Add when the culture is at log phase.

1. Measure the OD of the samples, for now using the induced cells and the BL21DE3 control
   1. OMPA1 + his = 0.93A
   2. OMPA1 + strep = 1.23A
   3. OMPA2 + his = 1.19A
   4. OMPA2 + strep = 1.34A
   5. BL21DE3 = 1.39A

Magnetic Labeling with MACS (6/28/07)

1. Create Standard MACS Separation Buffer:
   1. Dilute MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution (used 100 ml Rinsing solution with 5 ml BSA)
2. Followed Indirect Magnetic Labeling Protocol
   1. Note: OMPA1+Strep and OMPA1+His had low cell count
3. Then follow Magnetic Separation

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