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Team:TU Delft/protocols/restriction enzyme digestion
From 2010.igem.org
Restriction enzyme digestion
Materials:
- plasmid DNA or PCR product
- restriction enzymes (Roche and BioLabs)
- buffer (10x)
- H2O
- water bath at 37 °C
- heat block or water bath at 80 °C
Protocol:
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
Reaction for one sample:
| DNA | x μL (up to 1,0 μg) |
| Buffer (10x) | x μL (for 1×) |
| Restriction enzymes | x μL (10 units/μg DNA = 1 µL) |
| H2O | x μL |
| 20-25 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): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C
Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA
