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Team:TU Delft/protocols/restriction enzyme digestion
From 2010.igem.org
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Buffer H (Roche): 0.5 M Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 | 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 | ||
Revision as of 20:50, 19 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) | 2,0 μL (for 1×) |
| Restriction enzymes | x μL (10 units/μg DNA = 1 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 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
