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
13. Wash with 10 mL RSB250-A two times a. RSB250-A must be cold b. Collect wash fractions for later gel on ice 14. Elute with 1% of culture volume of RSB250-B a. Collect elution fraction on ice 15. Eluted protein can be stored at 4°C or -80°C

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