Team:TU Delft/protocols/restriction enzyme digestion

From 2010.igem.org

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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.
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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.

Revision as of 06:37, 20 July 2010

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