3 replies to this topic

[mayak]

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.

[bob1]

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.

[Nayeem991]

Which vector are you using? If there is any unique restriction site inside the amp^r 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 kan^r gene (blunt end PCR product would be good choice) at that side. I think that will work.

[mayak]

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.