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How to transform the cells witht wo different plasmids


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#1 anonymous

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Posted 30 January 2001 - 10:00 PM

I am planning to transform E.coli BL21DE3 cells with two different plasmids with two different antibiotic resistance (ampicillin and kanamycin). I wanted to know if there is a specific protocol I have to follow different from the protocol for transformation with one type of plasmid. If anybody has experience in this type of experiment, any suggestions are highly appreciated.

#2 anonymous

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Posted 30 January 2001 - 10:00 PM

No difference. Just do one at a time, i.e. transform one, then make competent cells of it (but with the necessary antibiotics added of course), then transform with the next one (two antibiotics used for selection). If you find this too troublesome, you can skip the making competent cells step as there are protocols for transforming cells straight from plate (altough the transformation efficiency will be lower).

#3 anonymous

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Posted 31 January 2001 - 10:00 PM

You are wrong of course. E.coli can be transform with two different plasmids. The main thing I believe is that the replicon should be different. A cell with one plasmid can happily be made competent and accept another plasmids.

#4 anonymous

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Posted 31 January 2001 - 10:00 PM

Well, according to my knowledge, it's impossible to transform one bacterial with two plasmid. Not like transfection in which cells can take up more than one plasmid (normally they take a few plasmid, either same type or different type), transformation is a process that the bacterial cells take one plasmid each. After it takes one, it lose the abilty to be transformed, even if you try to prepare the competent cell from that clone. Thus, don't waste your time on that. I don't know what the purpose is, but I believe there will be an alternative design.

Good luck!





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