I already tried different osmotic shock protocols, but it didn't work out (including the use of polymyxcin B sulfate).
Hi Thomas.
In our lab we've always used osmotic shock for PPF preparation from E. coli BL21 (DE3) pLysS. It always worked fine.
The method we use is described in the paper I attached to this answer. It's one of my lab's papers. As you can see we use this method for about 10 years now.
Hope this helps.
Hi Mario,
thank you for your answer. It might be that the pLysS makes BL21 more sensitive to osmotic shock. We tried a protocol similar to your, but without success. Do you apply any changes when working with plain BL21(DE3)?
Well, I can't help you on that one. I never worked with a non-pLysS strain. But maybe you're right and pLysS does help.
If it's not working and nobody can give a better solution, maybe you can try using a pLysS strain.
Hi Matthieu,
thx for your protocol. I would like to give it a shot, but do you can you help me with the ODU you usually get in your pellet. Would be great to adjust the volume :)
If BL21(DE3) does not work out, try JM109(DE3) - I found it to work better. With BL21(DE3) I used to get contamination with chromosomal DNA, JM109(DE3) internal membranes appeared more stable.
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