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Restriction Digest

Materials

  • ddH20
  • DNA sample on ice
  • Appropriate 10X buffer (see below) on ice
  • 10X BSA on ice (if necessary; see below)
  • Restriction enzyme(s) (see below) on ice
  • CIP (keep in freezer until needed)

Recipes

15 μL reaction

  • 5 μL DNA
  • 1.5 μL appropriate 10X buffer
  • 1.5 μL 10X BSA (if needed—see below)
  • Restriction enzyme(s) (see specific enzymes below for exact amounts)
    • Don't immerse the tip, draw from the surface to avoid excess enzyme
  • Add ddH20 to bring the final volume to 15 μL

Note: Use this recipe when verifying plasmid inserts
Note: You can check this reaction after 1 hour

30 μL reaction

  • 12 μL DNA
  • 3.0 μL appropriate 10X buffer
  • 3.0 μL 10X BSA (if needed—see below)
  • Restriction enzyme(s) (see specific enzymes below for exact amounts)
    • Don't immerse the tip, draw from the surface to avoid excess enzyme
  • Add ddH20 to bring the final volume to 30 μL

Note: Use this recipe when your DNA concentrations are sufficient
Note: For a BamHI–XbaI double-digest, use 3.3 μL of 10X buffer

40 μL reaction

  • 20 μL DNA
  • 4.0 μL appropriate 10X buffer
  • 4.0 μL 10X BSA (if needed—see below)
  • Restriction enzyme(s) (see specific enzymes below for exact amounts)
    • Don't immerse the tip, draw from the surface to avoid excess enzyme
  • Add ddH20 to bring the final volume to 40 μL

Note: Use this recipe when your DNA concentrations are low
Note: For a BamHI–XbaI double-digest, use 4.3 μL of 10X buffer

Procedure

  • Combine ingredients for recipe
  • Incubate at 37°C for 2.5 to 8 hours
  • If preparing a ligation, add 0.5 μL CIP to the vector 30 minutes prior to ending the reaction
  • When the reaction is complete you can store your samples in the -20°C freezer or proceed to  in-gel ligation

Enzymes

1Boldface indicates the preferred buffer for the enzyme.

Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.

Double Digests


Suggested Buffers

1Sequential digest recommended.
2We actually prefer to do the double-digest and use Buffer 2 for the BamHI–XbaI double-digest.
Note that a double-digest of Bam HI and Xba I is not recommended. However, by following the procedure above you won't have any problems.

Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.