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Orientation of a cloned DNA fragment (gene) in a plasmid vector can be checked by
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The usual PCR technique for fragment orientation involves one primer specific of the vector (upstream or downstream of the cloning site) and on primer within the cloning sequence. If the primer set is correctly oriented, PCR will give a unique band with a specific size according to the distance between primers. If not properly oriented, there will be no amplification. For digestion, principle is the same : using one restriction enzyme specific of the vector (a unique site close to the cloning site) and one restriciton specific of the insert but not lying right in the middle of it (it may be the same restriciton enzyme specific of the vector). You then may calculate the distance between the two cleavage sites according to insert orientation relative to the vector. As the site lying within the insert is not right in the middle of it, digest is generating two different fragment sizes according to insert orientation.