Endy:Restriction Digest

From OpenWetWare
Revision as of 09:12, 20 September 2007 by Barry Canton (talk | contribs) (Digest Mix)
Jump to: navigation, search


  • Prepped DNA. For a BioBrick assembly (and possibly any cloning operation) 300 ng is more than enough.
  • Restriction buffers (also from New England Biolabs. source)
    • For all combinations of the four BioBrick enzymes, we use Buffer 2 + BSA.
  • ddH2O

Digest Mix

  • 0.2-1μg DNA from prep or PCR
  • 10% (by volume) Restriction Buffer
  • 1% (by volume) BSA stock
  • 2.5% - 5% (by volume) of each restriction enzyme.
  • ddH2O to produce correct percentages by volume
  • Enzymes < 10% of total volume so glycerol is < 5% of total volume.

We prepare a restriction digest supermix (stored at -20C) containing -

  • 5*n μl of Buffer 2
  • 0.5*n μl of BSA
  • 37*n μl of ddH2O

where n is the desired number of 50μl reactions.

Example - 50μl digest

  • 42.5μl of restriction digest supermix
  • 1-5μl of preppedDNA
  • 1μl of each restriction enzyme

Reaction conditions

  • Incubate reaction at 37°C for 1 hour. Heat kill enzymes at 80C for 20 min.
  • Store at -20°C.


  • On improving the efficiency of EcoRI/SpeI Double Digest.
  • At glycerol concentrations > 5%, star activity is sometimes observed (meaning that the enzyme cuts at places other than its recognition site).