Double restriction enzyme digest - (Aug/23/2007 )
Hi, Hope somone can help me with my questions.
i intend to do as double enzyme digest on a plasmid for sub cloning.
My 2 sites is about 17bp apart, are this two sites too near from each other?
Any one knows where i can read up more on RE, i have read the web content from NEB.
Thanks a lot
you can digest your plasmid with double enzyme though it is 17 bp apart
Yeah, I suppose that as long as your restriction sites aren't so close that one digestion ruins the binding site for the next enzyme (I don't think this happens in a MCS) you won't have any problems. 17pb is ok.
Any molecular cloning (and molecular biology) book in general will have a chapter dedicated to restriction enzymes. Usually it's one of the first chapters because it's imperative to know how they work to understand molecular cloning in more detail.
Other than that, I suppose that if you google a bit you'll find some sites dedicated to the subject. Of course that a book is more adequate and reliable, but the net can give you some good hints.
i use a protocol similar to a sequential digestion which you find here
the method in theroy minimizes the reciprocal disturbing of enzymes on DNA