Figure 1. Mechanisms of EMP and RF cloning.

Mechanistic details of EMP and RF cloning are compared. (A) EMP cloning involves two PCR reactions. In the 1^st EMP PCR a forward primer F1 and a reverse primer R1 with overhang exponentially amplify the insert of interest. In the 2^nd EMP PCR reaction the purified product is used as a megaprimer to exponentially amplify the target plasmid together with a forward primer F1 and reverse primer R2. (B) RF cloning also includes two PCR reactions. In the 1^st RF PCR two overhang containing primers F1 and R1 exponentially amplify the insert of interest. In the 2^nd RF PCR the purified product is used as a megaprimer to linearly amplify the target plasmid. Experimental procedures are described in detail in Materials and Methods and Tables S1 and S2.