Please help with E.coli culture! - (Jun/29/2008 )

Hi, everyone,

Recently, I am desperate for bacteria culture. Before, I grew the bacteria expressing my protein very well. But recently, I always failed to grow bacteria expressing the same protein (the bacteria are from different batch, we usually do transformation before we need to express our proteins. we don't make glycerol stocks). They died before i induce. We thought it was because of phage contamination. So we repurified DNA and did transformation again. For some reasons, bacteria grow before we induce.But after induction, we can see something like lysates in flasks. When we spin the culture, no cells but some maybe debris.

Does anybody meet this situation? Does anybody know what is the reason? Please help me!

Thanks for your response in advance!

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Sounds like a toxic protein. Have you resequenced?

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Hi, Swanny,

Thanks for your comments! I will resequence it.

It is so strange that when i make BL21 competent cells, the same thing happened. Does anybody know why?

Thanks a lot in advance!

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Do you always make your own competent cells? If so, how old are the current ones? Are they the same batch as the ones you used initially? When were the reagents for preparing he cells made? Are they contaminated?
Can you get cells from someone else in your building? Can you buy in some competent cells?

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Hi, Swanny,

Thanks a million for your reply! From your suggestions, we borrow competent cells from others. They grew very well. So maybe the problem was that competent cells got contaminated.

Thanks swanny again!

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