Replacing the CMV promoter of a plasmid with another promoter
Posted 23 August 2011 - 10:19 PM
- Tona Que Hay likes this
Posted 24 August 2011 - 11:06 AM
...see this [url="http://www.biotechniques.com/BiotechniquesJournal/2010/June/Overlap-extension-PCR-cloning-a-simple-and-reliable-way-to-create-recombinant-plasmids/biotechniques-280116.html"]link![/url]
Posted 25 August 2011 - 05:45 PM
Another more traditional alternative is to use PCR to amplify the promoter of interest to give it the desired restriction sites.
(you can add RE site to the 5' end of primer - remember to add ~6bp to flank the restriction site. The enzyme needs space to bind on)
This amplified promoter (with your desired RE site) can then be ligated into the vector.
You can also do the same trick on the plasmid. If useful sites are unavailable you can use PCR to amplify the vector. Have one primer bind down stream of the old promoter and the second up stream of the old promoter. PCR amplify and you have removed the old promoter.