Lysis Protocol for E. Coli

Revised: 08/01 CHP

Summary of final step of previous procedure

• Take OD₆₀₀ before cfg
  
• Resuspend to an OD₆₀₀ of known amount or 10 ml /bottle of "buffer B" pH 8 (lysozyme is more efficient at pH 8.) and detergent
  Detergent: 40 mM octyglucodis (may cause aggregate in some mutannts or 0.1% triton X-100 ASR uses)
  

  50 mM Tris HCl pH 8
  25mM NaCl
  2mM EDTA
  

• I extract the pellet from the wall of the bottle and vortex till in solution or put in the cold room and shake on low for 5 to 10 min.
• Freeze sample

Lysis

• Make fresh Lysozyme (10mg/ml ) add 1/10 vol., ensure the buffer has EDTA, it is needed to help the lysozyme act

  --Add 1 ml of PMFS (phenyl methyl sulfonyl fluride sigma P-7626 ) 100 mM which is in DMSO or isopropanol, it is a serine protease and is not soluble in water. Be careful not to get on you.

  --or if a problem with serine proteases, try Roche complete protease inhibitor, one of the cat # 1-697-498

  --WHY? a protease inhibitor (while native tailspike is not sensitive to proteases, if you are examining the whole cell lysis you want to see all the tailspike chains [aggregates and native]) we need to preserve the intermediates.

• Shake at 30^oC or 37^oC in water bath (water is better at temp. tranfer) . 30 min
  
• Freeze in "liquid N₂₁" or back in the freezer, then thaw by incuabted at 37^oC about 15 min
  
• It will be very viscous from DNA, thus we need to add DNase 1mg/ml - 1/10 volume and 1M MgSO₄ 1/10 vol. is needed for the DNase to act , shake about 15-30min.

Take sample

• Cfg 9K 25 min GSA take sample.
  10 K 10 min SS34
  5min in the epindorf

* Resuspend the pellet of buffer B, nothing added, to see what is in it.