Engineering Enzyme Site - (Mar/25/2007 )
I want to engineer a unique enzyme site blunt end into my gene and one sticky end of my own interest. can any body give me some tips which steps i should have to consider while engineering enzyme site. My gene is eukaryotic origin and how can i be sure that the site which i will put is affected by mythylation or not.
where is this site? Is it within the gene or outside the gene?
A good places to start looking for for methlyation problem data wil NEB's website. The technical section on restriction enzymes. Always remember to give enough bp to flank the restriction site. NEB also gives this in on restriction enzyme efficient close to DNA ends. The enzymes have a foot print and they need to stand on something while they cut your restriction site.
I would need more info if you want suggestion for a cloning strategy.
Best of luck