Endy:Restriction Digest

Materials

• Need roughly 3-5 μL of prepped DNA. Aim for 1 μg of DNA.

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

• Restriction buffers (also from New England Biolabs. source)
  • EX – EcoR I & BSA
  • ES – EcoR I
  • XP – 3 & BSA
  • SP – 2 & BSA
  • ddH₂O

Notes for improving efficiency of EcoRI/SpeI Double Digest

Digest Mix

• μg 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. At higher glycerol concentrations, star activity is sometimes observed (meaning that the enzyme cuts at places other than its recognition site).

Example - 20μl mix

• 2.0 μL Restriction Buffer
• 0.2 μL BSA stock
• 3-5 μL DNA from prep or PCR
• 0.5 μL Restriction Enzyme Stock
• X μL ddH₂O (to bring solution up 20μL)

Incubate reaction at 37°C overnight (minimum 4-6 hours).

Store at -20°C.