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) 4,0 μL (for 1×)
Restriction enzymes x μL (10 units/μg DNA = 1 µL)
H2O x μL
40 μL


Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 65 °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

1x Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithiothreitol, pH 7.9

1x Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithiothreitol, pH 7.9

Note that the enzymes of BioLabs sometimes 0.01 mg/mL BSA require