Restriction digest of plasmid DNA
| Materials |
|---|
| Restriction enzyme stock (–20°C) |
| Appropriate 10x enzyme buffer (–20°C) |
| Sterile dd H2O |
| Depending on the digestion (optional):
BSA (bovine serum albumin): To ensure 100% activity of the enzymes, 100 µg/ml BSA should be added (stored at –20°C) |
Procedure for analysis purposes
- 300 ng of DNA samples (e.g. control digest of plasmid minipreps)
- 1 µl appropriate enzyme buffer (10x)
- 0.1 µl restriction enzyme 1 (1 U)
- 0.1 µl restriction enzyme 2 (1 U)
- opt. 1 µl BSA (10 mg/ml stock solution)
- Fill up to 10 µl with ddH2O
- Incubation for 3 h at recomended temperature
Procedure for preparative purposes (Overnight digestion)
- 2 – 4 µg of DNA sample (e.g. plasmid for preparative procedures)
- 2 µl appropriate buffer (10x)
- 0.2 µl restriction enzyme 1
- 0.2 µl restriction enzyme 2
- opt. 2 µl BSA (10mg/ml stock solution)
- Fill up to 20 µl with dd H2O
- ON incubation at recommended temperature