Standard Protocol for WarmStart® Nt.BstNBI (NEB #R0725)
Set up the following reaction on ice; if preferred, reactions can be set up at room temperature.
(Please note that enzyme should always be added last):
COMPONENTS 50 µl RXN FINAL CONC. OR AMOUNT DNA 1 µg
5 µl 1X Restriction Enzyme 1 µl Varies(1) Nuclease-free Water to 50 µl
We recommend 5-10 units of enzyme per µg of plasmid. Adjust according to protocol at NEBcloner.neb.com. For convenience, 1 µl can be used.
Gently mix the reaction by pipetting up and down and microfuge briefly.
Incubate at 55°C for 60 minutes.
Heat inactivate at 80°C for 20 minutes.