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