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 .
