The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. DpnI digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to DpnI digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during DpnI digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli. cells. The nicks will be repaired after transformation into the bacteria.