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Kan resistant plasmid growing in Amp

Kan resistant plasmid growing in Amp - Protocols and Methods Forum

Kan resistant plasmid growing in Amp - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 03-02-2005, 10:39 PM
Kyle Legate
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Default Kan resistant plasmid growing in Amp



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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Old 03-02-2005, 11:09 PM
Sergio
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Default Kan resistant plasmid growing in Amp

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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