NEBuilder HiFi DNA Assembly Reaction (E2621)
Set up the following reaction on ice (to 20μl total volume): 
Recommended Amount of Fragments Used for Assembly2–3 Fragment Assembly*4–6 Fragment Assembly**Positive Control✝Recommended DNA Ratiovector:insert = 1:2vector:insert = 1:1 Total Amount of Fragments0.03–0.2 pmols*X μl0.2–0.5 pmols**X μl10 μlNEBuilderHiFi DNA Assembly Master Mix10 μl10 μl10 μlDeionized H2O10-X μl10-X μl0Total Volume20 μl✝✝20 μl✝✝20 μl. 
Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled).
Following incubation, store samples on ice or at –20°C for subsequent transformation.
Transform NEB 5-alpha Competent E. coli cells (provided in the cloning kit or purchased separately from NEB) with 2 μl of the assembled product, following the transformation protocol.
