Re: Mutating CMV promoter

Posted by Miryan on Jan 25, 2002 at 09:35
poste8-188.crchul.ulaval.ca (132.203.188.8)

Re: Mutating CMV promoter (Thermus aquaticus)

Thanks Thermus aquaticus!

The gene I have to clone is already in an other vector between NdeI and XhoI sites (and the NdeI site contains the ATG of my protein). I want to digest with those 2 enzymes, and than ligate it in my new vector. BUT, this new vector contains the NdeI site in it's promoter. So, I want to remove this NdeI site, and than insert a linker into the multiple cloning site. This linker will contain the NdeI site... It is really the easiest way for me to do that cloning, and the alternatives are really risky and are not worth the shot... It's a tough gene to clone and to amplify by PCR (probably due to a real strong secondary structure), so I want to minimize the steps to do it. That way is the easiest way by far. About site directed mutagenesis, I've done that in the past, and I did not have any problem. It worked on my first try, so I am not worried. Thank you.

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