I need to prepare a bacterial lysate of mycobacterium where liquid culture contains 10^8-10^9 cells per mL. To increase yield I sonicate 15 mL at once. I tried different regimens of sonication, however, there's still growth on agar (which I use to control for sterility). To clarify, I need a whole cell lysate (not a protein) where cells are killed but not degraded too much (to preserve as many structures as possible) without adding any additional lysing substances. Are there any ideas of such protocol, since I failed to find one?
In brief, I need killed (but not totally destroyed) culture which would be sterile after sonication.
Hi there,
Filtration of the lysate on 0.2µm membrane provided the lysate is not too viscous.
Thank you! The problem with 0.2 filtration is that lysate is really viscous even after 5um filtration and clogs the 0.2 filter in a second.
As for antibiotics they also can not be used. I need pure lysate without any added substances for further experiments, except NaCl which is used to resuspend cells after centrifuge.
As I've mentioned, I can't use dnase or other substances. However, I think of three freeze-thaw cycles at –80°C, then lysis, then centrifugation to pellet whole bacteria. However, I have doubts about centrifugation RPM to avoid sedimentation of large particles (such as membrane debris) which are crucial for further experiments.
M tb is sensitive to heat and you can kill suspensions at 80°C for 20 min without affecting DNA. See doi: 10.1136/jcp.55.10.778
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