Standard Protocol for WarmStart® Nt.BstNBI (NEB #R0725)
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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.
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Gently mix the reaction by pipetting up and down and microfuge briefly.
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Incubate at 55°C for 60 minutes.
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Heat inactivate at 80°C for 20 minutes.