Standard Protocol for WarmStart® Nt.BstNBI (NEB #R0725)

  1. 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
    NEBuffer™ r3.1
    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.

  2. Gently mix the reaction by pipetting up and down and microfuge briefly.

  3. Incubate at 55°C for 60 minutes.

  4. Heat inactivate at 80°C for 20 minutes.