From 2011.igem.org

Digestion Protocols

Double Digestions

4 hour incubation (37°C)

We used serial digestions to improve SpeI efficiency since it wasn’t working correctly as follows:

We then purified the digestions using the Promega ® Wizard Sv and PCR Clean-Up system which were then eluted to 17.6 uL of ultrapure water or precipitated using Sodium Acetate and ethanol in ice.

[*] Plasmid opening [**] Fragment extraction

Run a 1% agarose electrophoresis for 55 minutes with 70V, and purify the expected weight bands using the Promega ® Wizard Sv and PCR Clean-Up system.