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Restriction Site Destruction

Cloning Restriction Blunt Klenow Digestion

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#1 Daniel Doro

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Posted 27 December 2011 - 04:10 PM

Greetings,
I need to double digest a gene (700bp) out of a plasmid (pet9a24) flanked by the enzymes Xba1, Xho1, which are exactly the sticky ends I need to clone into another plasmid. However, the plasmid has this Not1 site before Xho1 that I have to eliminate for further reasons.

Is it possible to destroy Not1 site by single digesting with Not1 and performing using Klenow (Large Fragment) to blunt the sticky-ends and then re-ligate it?

Will that eliminate the Not1 site?

Best regards,

Daniel Doro

#2 phage434

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Posted 28 December 2011 - 06:37 AM

It would (in my opinion) be far easier to use PCR to amplify your vector with 5' restriction sites of your choice, and choosing the primers to eliminate the NotI site. If you add NotI to the ligation reaction following PCR, you can even select for the construct you want.





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