IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 2

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Contents

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

  1. Take out control 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.

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. Used 200 ul of cells
    2. Note: OMPA1+Strep and OMPA1+His had low cell count
  3. Then follow Magnetic Separation

Results from MACS plates

Observation: There were many colonies that grew on our MACS assays, which was desirable. However, on our control plates, which we did not expect to grow, there seemed to be the same amount of growth.

Conclusion: There seems to be a lot of background binding taking place, involving contamination or infrequent wash steps during the MACS column phase.


Liquid Culture of Perry's Cherry Bacteria (6/29/07)

  1. Add 2 ml LB
  2. Add 2 ul Kan
  3. Add a colony of the bacteria using sterile technique (toothpick method).
  4. Incubate
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