orientation in cloning - (Oct/08/2007 )
can any one tell about orientation cloning
how to make sure that the insert cloned is in right orientation with vector
thanks in advance
i welcome ur great suggestions
Just do a digestion with and enzyme that cuts inside your fragment (not in the middle of it!) and another one that cuts in your vector.
Depending on the orientation of the fragment you'll get different bands when you run the whole thing on a gel.
Another way is to sequence it with a primer that anneals outside the fragment. Many plasmids have sites for universal primers like M13.