Endy:Restriction Digest

Materials

• Prepped DNA. For a BioBrick assembly (and possibly any cloning operation) 300 ng is more than enough.

• Restriction enzymes (EcoR I, Spe I, Xba I or Pst I)

• 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).