General Digestion Protocol

1.Digest Upstream Part with EcoRI-HF™ and SpeI.

Add 500 ng of plasmid DNA, EcoRI-HF (1 µl), Spel (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

2.Digest Downstream Part with XbaI and PstI.

Add 500 ng of plasmid DNA, XbaI (1 µl), PstI (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

3.Digest the Destination Plasmid with EcoRI-HF and PstI. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

Add 500 ng of plasmid DNA, EcoRI-HF (1 µl), PstI (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

4.Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Source

Thanks to NEB for the above instructions. The original source can be found here: [http://www.neb.com/nebecomm/products/protocol445.asp]