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Swich selection in plasmid

plasmid antibiotic cloning

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3 replies to this topic

#1 mayak

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Posted 12 July 2012 - 01:01 AM

Hi all,

I hope someone can help me. I have a gene I'm interested in on a plasmid. This plasmid has the amp resistent gene, but I want it to have the kan resistent gene. I don't want to transfer the gene to a new plasmid because I want the control system I have now, so I simply want to change the amp resistent gene to a kan resistent gene. What would be the easiest way to do that?

Thank you all,

Maya.

#2 bob1

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Posted 12 July 2012 - 01:07 AM

Sub-cloning like you would for the gene itself. I have to warn you though it will be easier to find a plasmid with the same promoter and subclone the gene.

#3 Nayeem991

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Posted 12 July 2012 - 04:46 AM

Which vector are you using? If there is any unique restriction site inside the ampr gene of your vector, just digest the vector with that enzyem...make it blunt ended by klenow fragment or T4 DNA pol and insert blunt ended kanr gene (blunt end PCR product would be good choice) at that side. I think that will work.

#4 mayak

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Posted 12 July 2012 - 05:59 AM

Thank you for your replies. I looked for restriction sites but I can't take the kan out of the plasmid, in any good way. I think bob1 is right, I will try to move the gene to a different plasmid and hope it will work. Thank you.
Maya.





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