Assembly utilizes two simultaneous enzymatic activities in a single reaction, Type IIS restriction endonuclease digestion and T4 DNA Ligase ligation. With optimized buffer components and enzyme ratios, a single reaction containing a destination plasmid and inserts (PCR amplicons or precloned) will result in ligation of inserts in the correct order and the accumulation of assembled product over time. The final assembly has none of the chosen Type IIS recognition sites, rendering the assembly inert to further digestion. For more information, view our online tutorial at www.neb.com/goldengate.