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Replacing the CMV promoter of a plasmid with another promoter

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#1 Discuss



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Posted 23 August 2011 - 10:19 PM

Need help with cloning. I want to replace the CMV promoter of a plasmid with a different promoter of interest. The plasmid contains my gene of interest under the CMV promoter.To replace the CMV promoter I planned to digest my promoter of interest from another plasmid and ligate into the CMV-plasmid. However among the two restriction sites I can find in the CMV promoter region of the plamid, one of them lies in the middle of the CMV promoter sequence and the other one after the promoter. How can I digest the entire CMV promoter region and replace it with my promoter of interest. The CMV promoter plus enhancer sequence is from 236-852 and the restriction site is at 484. If I digest it at 484 and replace it with my promoter, will the replacement still work?

#2 pDNA



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Posted 24 August 2011 - 11:06 AM

try restriction free cloning if you have no suitable restriction sites available:
...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]


#3 perneseblue


    Unlimited ligation works!

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Posted 25 August 2011 - 05:45 PM

Interesting use of PCR there pDNA. Nice find.

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.
May your PCR products be long, your protocols short and your boss on holiday

#4 Discuss



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Posted 25 August 2011 - 07:44 PM

Thank you both!!

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