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double transformations


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#1 deespike

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Posted 09 January 2011 - 05:22 AM

HI all

I have been facing a strange problem . I am getting 2 different plasmids having the same ori of replication but different antibiotic resistant genes(Amp and Kan) in the same host cell. The size of both the plasmids are different -12kb and 2.8kb . I get these 2 plasmids on gel after isolation under kanamycin selection. I am not able to eradicate the amp resistant contamination as it lowers the other plasmid yield after subsequent subculture also.

Plz give me some suggestions for coming out of this problem.

#2 phage434

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Posted 09 January 2011 - 08:22 AM

Take your isolated DNA (or better, a gel isolated sample of the band you want) and re-transform into your strain. This will be easier than trying to cure the plasmid. If you really need to cure it, then EtBr exposure or high temperatures can sometimes work. You'll probably need to do replica plating on both Kan and Amp plates to find colonies with only Kan resistance. Re=transformation will be much easier.




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