Team:Penn/Protocols

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Penn 2012 iGEM Wiki

Protein Purification

RSB250-A Recipe:

  • 20mM Tris
  • 250mM NaCl
  • 30mM Imidazole
  • 10% Glycerol (v/v)
RSB250-B Recipe
  • 20mM Tris
  • 250mM NaCl
  • 500mM Imidazole
  • 10% Glycerol (v/v)

Induction

  1. Grow a small booster culture in LB Broth overnight (~2-10% of final culture volume) w/ appropriate antibiotics
  2. Innoculate full scale culture & monitor OD600 every 15-30 minutes until OD600 reaches 0.8
  3. At OD600=0.8, induce with appropriate concentration of inducer (e.g. 1mM final concentration of IPTG for lac promoter)
  4. Allow to grow overnight @ 25°C

Pelleting & Sonication

  1. Collect cultures by centrifugation for 30 minutes @4°C & 5000g
    • If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)
  2. Resuspend pellet in RSB250-A (minimum 5% of culture volume)
  3. Pellet must be completely dissolved (no cell clumps) for efficient lysis.
    • For especially difficult to resuspend pellets, sucking up and ejecting the cell suspension through a large gauge (narrow) needle several times can break up the pellet effectively
  4. Sonicate 5 cycles
    • 1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down
    • Lysis must be done on ice or excessive protein degradation will occur
  5. Spin lysate down @4°C & 20000g for 30 minutes
  6. Transfer supernatant into 15 mL Falcon tubes
  7. Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)
    • Wash beads 2-3 times with equal volumes of RSB250-A
    • Spin down cells @ ~3000 rpm in a microcentrifuge
  8. Rotate overnight in a 4°C cold room
  9. Pour lysate and beads into a chromatography column, allow to empty through gravity flow
    • Retain flow through (FT) on ice for later analysis
  10. Wash with 10 mL RSB250-A two times
    • RSB250-A must be cold
    • Collect wash fractions for later gel on ice
  11. Elute with 1% of culture volume of RSB250-B
    • Collect elution fraction on ice
  12. Eluted protein can be stored at 4°C or -80°C

Thawing Mammalian Cells

  1. Prepare plates with 9mL of appropriate supplemented cell culture media
  2. Take the cells out from the -80 freezer
  3. Shake cells in a water bath at 37 degrees C