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Tet-On3G: cutting CMV promoter out of pCMV-Tet3G

molecular cloning tet-on

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#1 dsjensen

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Posted 12 December 2013 - 04:38 AM

Hi,

 

I'm going to use the Tet3G coding sequence from pCMV-Tet3G and link it to another promoter.

Do anybody have experience in excising the CMV promoter sequence from the pCMV-Tet3G plasmid - and replacing it with another promoter. And then transform it in competent cells and subsequently purify the plasmid DNA.

 

This way I can make the restriction sites match and do not need to do multiple subclonings steps.

 

Thanks



#2 bob1

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Posted 12 December 2013 - 11:48 AM

The easiest way to generate compatible ends is to design primers with RE sites on the  5' end, then do a PCR of the site of interest, digest, and clone.







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