At the end of the day, all you want to know from a restriction screen is whether your insert is in the vector. But while the standard “chop out a fragment” approach favored by most researchers provides good information about the presence of the insert and its orientation, it only uses a portion of the potential power of a restriction screen.
It doesn’t take much more work to set up a restriction screen that is more informative, allowing you to confirm a negative result and the identity of your vector, at the same time as checking for your insert. Here’s a few ways you can soup-up your restriction digest.
1. To confirm negative results: If you screen by cutting out your insert using the enzymes you cloned in with, but don’t get any insert bands back it’s difficult to know whether this is a negative clone or just a dirty prep that didn’t digest properly. An alternative approach is to use restriction enzymes that cut in the vector itself and flank the multiple cloning site so that a negative result generates a fragment, but a positive clone will generate a bigger fragment.
2. To test positive results: If you want a quick test of whether your insert has actually been cloned in, a good approach is to chop out a fragment from the insert itself. This way you can simultaneously verify the presence of an insert and its identity. This can be especially useful if the cloning-in restriction sites have been damaged during cloning.
3. To confirm the integrity of the vector: Vector deletions (thanks to E. coli) can occur in GC-rich regions, and star activity can delete portions of the vector on the 5` and 3` side of the cloning site during vector preparation (especially when vectors are not well prepared – see proper vector preparation). Restriction screens can be designed to digest on both sides of the multi-cloning site and at different positions around the vector so that the integrity of the vector can be confirmed. After all, we want the vector to work too!
Let us know your restriction digest tips in the comments.
