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#1
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| Has anyone experienced this before? I can't find an explanation, and I would really like one. I am cloning in pEGFP-C1, which contains a Kan resistance cassette. I cut, ligated, plated, mini-prepped and maxiprepped, all using Amp by default because 90% of my plasmids encode Amp resistance. However, when I was making the data sheet for the new plasmid I discovered that it encodes Kan resistance, not Amp resistance. How could this be? How could a Kan resistant plasmid be amplified in Amp-containing media? I am sure I didn't accidentally use a Kan stock, and I am sure I have the correct product. I am not the only person who has experienced this; a colleague did the exact same thing (different Kan resistant plasmid) late last year. kyle |
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#2
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| To discard the trivial explanations, i.e. bad ampicillin/ampicillin resistant strain/contaminations/etc, I'd suggest to perform a control experiment transforming some cells with the vector (not your construction) and plating both in Km and Amp. If the vector is responsible for the amp resistance, you should get aprox same number of colonies in both plates, and zero colonies in the non-transformed cells plates. If so, you should consider what Clontech says about this vector: (copied and pasted) Note: The vector sequence file has been compiled from information in the sequence database, published literature, and other sources, together with partial sequences obtained by CLONTECH. This vector has not been completely sequenced. Maybe the vector still carries a functional bla gene from one of its parental plasmids.... Sergio |
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| amp , growing , kan , plasmid , resistant |
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