Lysin purification, comparison

Lysin purification - comparison

StepOld procedure (Jane Kennedy)New procedure
Sample volume500ml lysed cells20ml lysed cells
Pellet residue 5K, 5 min at 4C (cell residue only)25K, 20 min at RT (residue + virus)
Wash buffer500 ml BBM20ml, 0.1MLiCl, 25mM KOH, 50mM MOPS (pH 7)
Pellet residue5K, 5 min at 4C25K, 20 min at RT
Resuspension buffer125ml, 500 mM KCl, 20 mM Tris, 2 mM 2-ME, pH 7.52ml, 0.5M LiCl, 25mM KOH, 50mM MOPS (pH 7)
Pellet residue12K, 5 min at 4C25K, 20 min at RT


Changes:
1- Smaller volumes
2- Pellet both cell residue and virus
3- Omit reducing agent

Further improvements:
1- Add EGTA to buffers
2- Wash with higher salt (0.2M LiCl)??
3- Dilute and ppt DNA w/protamine sulfate