Team:Queens Canada/restriction

Restriction Digest of DNA · Benchling

Restriction Digest of DNA

Introduction

Restriction enzymes are naturally occurring bacterial endonucleases that recognize a large range of DNA sequences. Given the variety of these enzymes and the unique sites they recognize, restriction digests have become the most widely used method scientists employ to selectively move a specific piece of DNA from one plasmid to another. A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave DNA at specific sequences called restrictions sites. Often, the size of the plasmid insert and vector backbone are known and thus this technique can be quickly used to verify your plasmid.

Materials

  • DNA
    • Restriction Enzyme(s)
      • Appropriate restriction digest buffer (see manufacturer's instructions)
        • Buffer
          • BSA (if recommended by manufacturer)
            • dH2O up to total volume
              • Gel loading dye

              Procedure

              • Restriction Digestion
              1. Select restriction enzymes to digest your plasmid.
              1. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
              1. In a 1.5mL tube combine the following: DNA Restriction Enzyme(s) Buffer BSA (if recommended by manufacturer) dH2O up to total volume
              • example:
              • 1 µg DNA
              • 1 µL of each Restriction Enzyme
              • 3 µL 10x Buffer
              • 3 µL 10x BSA (if recommended)
              • x µL dH2O (to bring total volume to 30µL)
              1. Mix gently by pipetting.
              1. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions.
              1. Visualize results through Gel electrophoresis. Compare to known lengths