Double Digestion

1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.
Double Digestion

	Volume (μl)
Plasmid DNA	Up to 1μg
10×FastDigest Buffe	2
Enzyme1	1
Enzyme2	1
ddH2O	To 20
Total	20

2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 30 min.
4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)