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[joy123]

Hi, I want to do gene knockout in a G(+) bacteria. Now I have some difficulties with the bacteria transformation. This bacteria is seldom studied, and there is no report about how to transform vectors to this bacteria.



















I am using an electroporator for transformation. I have tried 1kV,2kV,3kV in 0.1cm cuvette, 4ms. It turned out that the 3kv kills the bateria. 1kV and 2kV keeps the bacteria alive, but the transformation failed.


I think transformation mainly depends on 2 aspects: 1. proper voltage and pulse time 2. the bacteria doesn't restrict the plasmid replication. I don't know if my understanding for bacteria transformaiton is correct, and I hope to get suggestions.



Do you have suggestions what I should do next? And how can I know if my plasmids (I have tried 3 plasmids) are resticted to the bacteria?

Thanks! And look forward to hear from your suggestions!