Buffer composition 50mM tris hcl
Nacl 150mM
Mgcl2 5mM
10%glycerol
1) Try leaving cell pellet in -80 C ON, this often helps
2) Cell wall degrading enzymes (would lysozyme work in your case?)
3) In yeast, reducing agents also often help to open the cell wall.
4) If it possible, make your buffer hyposmotic (No glycerol, no NaCl)
Are you sure your French Press is working properly? I have seen couple of papers that use French Press high pressure method to lyse B. megaterium. How did you decide that lysis wasn't succesful? French Press has the disadvantage that sample heat up quickly due to the compression. If you say you continued up to 2.5 hrs do you mean you left the sample for 2.5hrs under pressure? Cell disruption by French Press doesn't happen to the compression itself. Once the target pressure is reached you open a small spigot and you collect your sample in a tube. The rapid change from high pressure to normal pressure is what causes cell disruption. So leaving the sample under high pressure for 2.5hrs didn't add to the lysis process.
I Decide on the basis of microscope..cells are not aggregate.they move freely..as compare to enzymatic method i did to check the cell..
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