Rapid (Ligase-Free) Subcloning of PCR Products

The polymerase chain reaction (PCR) is a versatile, widely used method for the production of a very large number of copies of a specific DNA molecule (1 ,2 ). For some applications, it is advantageous to subclone the PCR product into a plasmid vector for subsequent replication in bacteria (3 –6 ). Subcloning the PCR product into a plasmid vector has several advantages: The amplified fragment can be sequenced with greater reliability, only one allele is sequenced per clone, and the vector containing the PCR product may be used for other molecular biological experiments, e.g., in vitro transcription, transfection, and further amplification in bacteria.