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QuikChange with Different plasmids

PCR mutagenesis

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#1 jas6cc



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Posted 20 September 2012 - 11:26 AM

I am trying to perform a Quikchange mutagenesis using PCR. I have been able to get this to work in the past but now I'm having some trouble.

I have been able to get a mutation to work using one plasmid, but I got another plamsid with the same sequence (only the protein is tagged with an epitope for purification) and the PCR mutagenesis does not work.

Any thoughts as to why the same mutagenesis works on the same protein sequence (I have confirmed it's the same sequence) in one plasmid but not work in a differen plasmid?


#2 Trof


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Posted 20 September 2012 - 01:11 PM

Well because plasmids don't care about protein sequence, they care about DNA sequence. What type of tag it is? Can it prevent amplification of a plasmid at current conditions, by adding a high GC region or something?
Are you sure that the kit is OK right now? (if you still use the old one) (dNTPS aren't degraded, ...)

And what exactly means "does not work"? You got no colonies, you got only wt, you can't see a product after PCR mutagenesist?

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