Team:BIOTEC Dresden/Protocols:Restriction Enzyme Digestion (Single digest)



Use as much DNA as possible (~5ug) for this starting step.

This way, you will have lots of DNA for ligation and electroporation after purification steps.


  • pure DNA (PCR product or plasmid)
  • DNA must be pure
  • DNA concentration must be known
  • 10X Buffer
  • Bovine Serum Albumin (100X)
  • Restriction Enzyme(s)
  • Ultra pure water


  • Calculate volume of DNA required to have about 5ug DNA
  • To a 1.5ml microfuge tube, add the following:
  • 5µL 10X Buffer (usually Buffer 4, but check)
  • 0.5µL 100X BSA
  • 1.0µL Restriction enzyme (20 Units)
  • Adjust final volume to 50µL with ultra pure water
  • vortex reaction mixture
  • pulse spin
  • incubate at 37°C for 1 hour
  • Hint: some form of purification step after the Restriciton digestion is required. Depending on your digested DNA, consider using the Qiagen kits for ‚PCR purification’ or ‚Gel Purification’. This step is essential to reduce background.
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