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

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m (Omp10mer and Omp 15mer Digestion and Ligation (7/13/07) =)
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===Omp10mer and Omp 15mer Digestion and Ligation (7/13/07) ====
===Omp10mer and Omp 15mer Digestion and Ligation (7/13/07) ====
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For Digestions, did 2 sequential digests, 1960 ng vector DNA (J04500), 951 ng insert DNA (Omp10mer and Omp15mer), using 1 to 3 molar excess ratio
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For Digestions, did 2 sequential digests, 1960 ng vector DNA (J04500)-980 for each, 475 ng insert DNA (Omp10mer and Omp15mer each), using 1 to 3 molar excess ratio
#Digested J04500 7 ul with 2 ul NEB3, 2 ul BSA, 1 uL Pst1, 8 uL H2O
#Digested J04500 7 ul with 2 ul NEB3, 2 ul BSA, 1 uL Pst1, 8 uL H2O
#Digested 10mer 6.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 8.2 uL H2O
#Digested 10mer 6.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 8.2 uL H2O

Revision as of 11:43, 13 July 2007

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

MACS with strep and his plus cherry/red bacteria (07/02/07)


Run the MACS with the cherry/red bacteria incorporated to see if separation occurs (07/03/07)

  1. Followed Indirect Magnetic Labeling Protocol
    1. Used 300 ul of Strep/His and 300 ul of RFP Bacteria
    2. Note: OMPA1+Strep and OMPA1+His had low cell count
  2. Then follow Magnetic Separation

Results from MACS plate

Observation: Still have some background in the elution fraction, instead of the plain white strep and his colonies that we expected.

Conclusion: We may need to further optimize the wash steps, because some of the extraneous bacteria expected to be gone appear in the elution fraction.


Transformation of BL21DE3 RFP cells (07/05/07)

  • For use in MACS assay.

pre-MACS work done by Mike (07/09/07)

  1. Grew overnight cultures
  2. 1-5 dilution
  3. Grew to 0.6-0.7 OD and induced with IPTG for 3 hours.

MACS Column for separation of red cells #2 (07/09/07)

  1. Followed Indirect Magnetic Labeling Protocol
    1. Used 300 ul of Strep/His and 300 ul of RFP Bacteria
    2. Used 8 ul his antibodies and 25 ul strep antibodies
    3. Used 30 ul of Beads
    4. Added an extra wash step after step #5
  2. Then follow Magnetic Separation
    1. Use the additional measures

Results from MACS plates and colony count (07/10/07)

Colony count after MACS:
OmpA1 + his = 13 white, 16 red.
OmpA1 + strep = 10 white, 110 red.

Observation: We still had background despite the many wash steps and additional beads.

Conclusion: The antibodies/beads don't seem to be binding properly, with no specific binding.


FACS Cell Sorting Culture Preparation (7/13/07)

  1. Prepared an overnight culture:
    1. OMPA1 + his
    2. OMPA1 + strep
    3. RFP-labeled cells

FACS Cell Sorting Preparation (7/14/07)

  1. Cell Innoculation and Induction
  2. Added antibodies according to the Fluorescent Labeling Protocol.
  3. Took the bacteria to the FACS cell sorter and sorted cells, plating them afterwards.

Omp10mer and Omp 15mer Digestion and Ligation (7/13/07) =

For Digestions, did 2 sequential digests, 1960 ng vector DNA (J04500)-980 for each, 475 ng insert DNA (Omp10mer and Omp15mer each), using 1 to 3 molar excess ratio

  1. Digested J04500 7 ul with 2 ul NEB3, 2 ul BSA, 1 uL Pst1, 8 uL H2O
  2. Digested 10mer 6.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 8.2 uL H2O
  3. Digested 15mer 10.8 uL with 2 uL NEB3, 2 uL BSA, 1 uL Pst1, 4.2 uL H2O
  4. QiaGen PCR Purification on all 3
  5. Digested J04500 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Spe1, 4 uL H2O
  6. Digested 10mer 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Xba1, 4 uL H2O
  7. Digested 15mer 30 ul with 5 ul NEB2, 5 ul BSA, 1 uL Xba1, 4 uL H2O

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