Team:TU Delft/protocols/restriction enzyme digestion


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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