Team:Queens Canada/Diagnostic-Restriction-Digest

Diagnostic Restriction Digest · Benchling

Diagnostic Restriction Digest

Introduction

The goal of a diagnostic digest is to cut your plasmid into specific sized pieces and analyze the resulting fragments by gel electrophoresis. The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert. By selecting the appropriate enzyme(s), one can either linearize a plasmid to determine the size of the entire construct or excise some or all of an insert from it

Materials

  • Liquid DNA aliquot of your plasmid of interest
    • Appropriate restriction enzymes
      • Appropriate restriction digest buffer
        • Gel loading dye
          • Electrophoresis buffer

          Procedure

          1. In a 1.5 mL tube combine: DNA, restriction enzyme(s), buffer, BSA (if recommended by manufacturer), dH20 up to total volume. Diagnostic digests typically involve ~500 ng of DNA.
          • *Example: 1 µg DNA, 1 µL of each Restriction Enzyme, 2.5 µL 10x Buffer, 2.5 µL 10x BSA (if recommended), x µL dH2O (to bring total volume to 25µL)
          • **Pro-tip**: The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut. By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour. Using this ratio, you can calculate the minimal amount of enzyme for your reaction. However, keep in mind that restriction enzyme activity is determined under ideal conditions with very clean DNA, so using a little more enzyme is advisable. Reactions are often performed with 0.2-0.5 µL of enzyme because it is difficult to pipette less volume than this; 0.2-0.5 µL will likely be more enzyme than you will need, but that's okay because a little more enzyme is usually better.
          • Store enzymes on Cooling Block!!!!
          1. Mix gently by pipetting
          1. Incubate tube at approximate temperature (usually 37°C) for 0.5- 1 hour.
          1. Conduct gel electrophoresis