Lysin purification, comparison
Lysin purification - comparison
Step Old procedure (Jane Kennedy) New procedure Sample volume 500ml lysed cells 20ml lysed cells Pellet residue 5K, 5 min at 4C (cell residue only) 25K, 20 min at RT (residue + virus) Wash buffer 500 ml BBM 20ml, 0.1MLiCl, 25mM KOH, 50mM MOPS (pH 7) Pellet residue 5K, 5 min at 4C 25K, 20 min at RT Resuspension buffer 125ml, 500 mM KCl, 20 mM Tris, 2 mM 2-ME, pH 7.5 2ml, 0.5M LiCl, 25mM KOH, 50mM MOPS (pH 7) Pellet residue 12K, 5 min at 4C 25K, 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