Griffitts:Restriction digest

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)

15 μL reaction
Note: Use this recipe when verifying plasmid inserts Note: You can check this reaction after 1 hour
 * 5 μL DNA
 * 1.5 μL appropriate 10X buffer
 * 1.5 μL 10X BSA (if needed&mdash;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

30 μL reaction
Note: Use this recipe when your DNA concentrations are sufficient Note: For a BamHI–XbaI double-digest, use 3.3 μL of 10X buffer
 * 12 μL DNA
 * 3.0 μL appropriate 10X buffer
 * 3.0 μL 10X BSA (if needed&mdash;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

40 μL reaction
Note: Use this recipe when your DNA concentrations are low Note: For a BamHI–XbaI double-digest, use 4.3 μL of 10X buffer
 * 20 μL DNA
 * 4.0 μL appropriate 10X buffer
 * 4.0 μL 10X BSA (if needed&mdash;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

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
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.