User:Matt Hartings/Notebook/Photosynthesis/2012/06/19

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Objective

Continue sperm whale myoglobin expression as started by Tamra notebook page and finish purification of protein as started yesterday notebook page.

Description

Start WT Hemoglobin Expression

  1. Inoculate starter cultures (35mL LB, autoclaved) with 35uL of 100mg/mL Ampicilin and cells from WT Ascaris Hemoglobin glycerol stock in -80 freezer.
  2. Shake/incubate the cultures overnight at 37C

Continuing Myoglobin Expression

  1. Spin down cells in starter culture (20 minutes, 4000 rpm, 4C)
  2. Prep expression culture media
    1. Add autoclaved phosphate to autoclaved TB with sterile technique.
    2. Add 1mL of 100mg/ml Ampicilin
  3. Innoculate expression cultures with resuspended cell pellets from starter cultures.
  4. Shake at 165rpm and 37C until OD reaches 6.6

Purification of WT Ascaris Hemoglobin

  1. Equilibrate Q column with water and then with Buffer A (25mM Tris, 50mM NaCl, pH 8)
  2. Put sample on superloop and inject sample onto column
    1. 200mL of sample. Adding to column will take longer than desired
  3. Run 25mL of Buffer A over column after injection finishes
  4. Start Eluting
    1. Buffer B (25mM Tris, 1M NaCl, pH 8)
    2. Gradient: 0-50% Buffer B in 30 minutes
    3. Collect Fractions

Data

  • Add data and results here...

Notes

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