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Comparative Physiology @ Guelph

New WSU TnI Pellet Protocol

Use freshly transformed BL21 cells!

  • Mid-afternoon, 4 L of LB + 30mg/L Kamamycin inoculated with 20
  •           Colonies in each 2L flask, then grow overnight 37oshaking
  • Take 1 ml sample of O/N growth for SDS Page analysis
  • Spin culture at 7,000rpm for 5 minutes to pellet, toss super
  • Resuspend pellets in 200ml Sonication Buffer
  • Sonicate 10-15 times on high power for about 1 minute-don’t foam
  • Let solution stay on ice while sonicating, let rest between bursts to cool
  • When finished, solution has consistency of water-test with pipet bulb
  • Rinse probe with dd water, EtOH, dd water again
  • Spin 10,000rpm 1 hour  Save super for column purification


Sonication Buffer-pH 8 at 4o        1 liter

6M Urea                                   360.36 g urea

50mM Tris pH8                      Â Â  50 ml 1 M Tris

5mM EDTA                               10 ml 0.5M EDTA

0.4mM PMSF                          Â Â Â  4 ml 0.1M

cocktail optional due to 6M urea