A rapid method to determine the orientation of blunt end ligated polymerase chain reaction products

Subcloning of polymerase chain reaction (PCR) fragments is often performed via blunt end ligation after previous Klenow fragment exonuclease treatment. Since insert-specific primers are at hand from the original amplification step, we used a simple PCR-based assay to determine the insert orientation of the recombinants. After purification of enriched plasmids, small aliquots were tested performing standard PCR reactions using a plasmid primer directed towards the cloning site and one of the insert specific primers, respectively. Only the distant insert primer yields a PCR product which can be visualized after gel electrophoresis. In this way both the insert orientation in the vector and the correct size of the cloned fragment is determined rapidly without sequencing or restriction fragment analysis.