i am trying to lysis the bacterial pellet (500ml culture pellet) with 50ml of lysis buffer ( 0.5 m Tris HCL, 5M NaCl pH 8.0, 100mM PMSF, Lyzozyme ) by sonication (Amp 20, Esp time 10 mins, Pulse on 15 SEC, Pulse off 10 sec ) but its not working. i have used the same method previously, it worked well. But facing problem for the first time. what might be the reason ? or any suggestions