Preparing bacterial protoplasts! - anyone has a good protocol? (Oct/08/2008 )

Hi there!

I want to make bacterial protoplasts of my poor B. subtilis cells. But I'm totally green on this area, does anyone have any good protocol for this?

Also, can you see if they are alive or dead? Morphology?

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Lysozyme would be a good enzyme to get rid of the cellwall, i really doubt they will still be alive though after the treatment. Might even expect some of them to lyse perhaps?!

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Lysozyme ok, but for how long? And in which buffer?

acctually don't care if they are that alive, I anyway want to kill them afterwards
but if they where whole would be an advantage.

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Try looking for plasmid prep from bacillus subtilis.

Even though it's not exactly what you ultimately intend to do but the first step involves stripping off the peptidoglycan wall!

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