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Restriction Enzyme Digestion: How does it work? Why is it useful?


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Restriction Enzyme Digestion: How does it work? Why is it useful?

In this activity related to plant biotechnology, learners use restriction enzymes to cut up DNA from a virus called Bacteriophage λ, a process known as restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. This lab will help learners understand what a DNA restriction enzyme is and how it works, how to use a micropipette, how to separate DNA using electrophoresis, and how to use a restriction digestion map to identify a sample DNA. This lesson guide includes background information, safety precautions and notes, and questions with answers for learners. For safety reasons, adult supervision is recommended. Modifications for younger learners are included in a related PDF (see related resources).

Quick Guide


Preparation Time:
1 to 7 days

Learning Time:
1 to 7 days

Estimated Materials Cost:
Over $20 per group of students

Age Range:
Ages 11 - adult

Resource Types:
Activity, Experiment/Lab Activity, Lesson/Lesson Plan

Language:
English

Materials List (per group of students)


  • Four microtubes
  • Microtube rack
  • 20-µl micropipette (or 10-µl micropipette) and sterile tips
  • Waterproof pen
  • Beakers or foam cups with crushed ice
  • 20 µl of 0.4 µg/µl λ DNA
  • 2.5 µl BamHI restriction enzyme
  • 2.5 µl EcoRI restriction enzyme
  • 2.5 µl HindIII restriction enzyme
  • 10 µl distilled water
  • Gloves
  • 500-ml beaker
  • Electrophoresis chamber
  • Power supply
  • 20 µl 10X loading dye
  • 1.0% agarose gel
  • Container with TBE solution
  • 37°C water bath w/ floating rack
  • 60°C water bath or saucepan on a hot plate
  • Cooler with crushed ice
  • Freezer (non frost-free, if possible)
  • Camera if desired
  • Distilled water
  • 0.002% methylene blue stain

Subjects


  • Engineering and Technology
    • Engineering
      • Bioengineering/Biomedical Engineering
    • Technology
      • Agriculture and Biotechnology
  • Life Sciences
    • Cells
      • Cell Structure and Function
      • Chemistry of Life
    • Diversity of Life
      • Viruses and Bacteria
    • Heredity and Genetics
      • DNA Structure and Function
      • Genetic Engineering
  • Physical Sciences
    • Chemistry
      • Chemistry of Life
  • The Nature of Science
    • The Scientific Process
      • Conducting Investigations
      • Gathering Data
      • Formulating Explanations
      • Communicating Results

Audience


To use this activity, learners need to:

  • see
  • see color
  • read
  • touch

Learning styles supported:

  • Involves teamwork and communication skills
  • Involves hands-on or lab activities

Other


Components that are part of this resource:

This resource is part of:

Access Rights:

  • Free access

By:

  • Stephens, Janice; Leach, Jan

Rights:

  • All Rights Reserved, The American Phytopathological Society, ©2011

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