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From 2010.igem.org

Restriction enzyme digestion

Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. Restriction enzymes we use are: EcoRI, XbaI, SpeI, PstI

Reaction for one sample:

DNA	x μL (up to 1,0 μg)
Buffer (10x)	2,0 μL (for 1×)
Restriction enzymes	x μL (5 units/μg DNA = 0.5 µL)
H2O	x μL
	20 μL

Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80 °C for 10 minutes and centrifuge shortly.

Used Buffers:

Buffer H (Roche): 0.5 M Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5