Protocol for insert orientation - (Mar/02/2006 )

Hi folks,
Can any1 hint me on designing a protocol for a colony on which i have same resitriction ends
of HindIII.

If you know the sequence of your insert and of your vector, find 2 enzymes, one that cuts your vector and one that cuts your insert and do a double digest. Put it on gel and look at the size of the fragments.
(first thing to do is a miniprep of you colony).

Also, another possibility is to do a PCR with one primer attaching to your vector and another one to your insert, this should give a positive result for good orientation, and negative for the other orientation.