Restriction Enzyme Digest(8/2004) 【University of Chicago】

Restriction enzymes recognize a specific sequence of nucleotides. Once they encounter that sequence, they cleave the DNA in a variety of patterns depending on the enzyme. They are used for a variety of purposes. One such purpose is gene mapping. In gene mapping CAPS (Cleaves Amplified Polymorphic Sequences) markers are often used to differentiate ecotypes.

1. Transfer 5µL of the DNA template (PCR products) to plate.

2. Prepare digestion cocktail according to following recipe:

With BSA*

ddH2O 10X Buffer 100X BSA* Enzyme PCR Product TOTAL

12.6µL 2µL 0.2µL 0.2µL 5µL 20µL per reaction

OR Without BSA*

ddH2O 10X Buffer Enzyme PCR Product TOTAL

12.8µL 2µL 0.2µL 5µL 20µL per reaction

*BSA (Bovine Serum Albumin) is needed by some enzymes to prevent the adhesion of those enzymes to reaction tubes and pipette surfaces. BSA also stablizes some proteins during incubation.


3. Allow digestion reaction to proceed for 1-2 hours (for mapping purposes) or overnight (for cloning purposes.)

4. Add 4µL 6X loading dye to sample(s) and run the gel with 12µL of each sample. NOTE: Differing gel concentrations may require different voltage and time settings.