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From 2011.igem.org

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Digestion Protocols

Double Digestions

2 hour incubation (37°C) followed by an hour incubation period at room temperature and another 2 hours at 37°C.

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

Ultrapure water	23.8ul
Promega ® Buffer D	4ul
XbaI	0.6uL
EcoRI*/PstI**	0.6uL
DNA	1ug
Final volume	40uL

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.

Promega ® Buffer H	2uL
EcoRI**/PstI*	0,6uL
DNA	17,6uL
Final volume	20uL

[*] 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.