Endy:Restriction Digest

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Contents

Materials

  • 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 at least 1 hour; longer digest gives more complete digestion, especially if you have >1µg DNA, but can at times give nonspecific digestion, even if glycerol is <5%. Heat kill enzymes at 80C for 20 min.
  • Store at -20°C.

Notes

  • 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).
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