PGem Easy Vector System: ampicillin conc and plasmid length problem - (Sep/20/2010 )

If you have a primer that binds to the vector and one that binds to the insert, always use that set if you're going to do PCR on colonies directly from the transformation plate. Alternatively, you can pick the transformants from the transformation plate to a fresh plate, grow that overnight, and do colony PCR using growth from the fresh plate as template. If you pick your transformants first, you can use either two primers that target the insert or one that binds to the vector and one that binds to the insert.

In either case, note that using a primer that binds to the vector and one that binds to the insert is orientation specific -- if you use a vector borne forward primer and an insert borne reverse primer, clones that contain the insert in the opposite orientation will not amplify. If the orientation of your insert is important, this can be a good thing -- you get both pieces of data (the presence of the insert and its orientation) simultaneously. If orientation of the insert is irrelevant, you'll miss some positive clones unless you do two PCRs on each. Using two insert-specific primers avoids this problem, but you must patch you colonies to fresh media first to avoid false positives.