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Basic Mechanics of Cloning: Restriction Enzymes & Cloning Vectors

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Session Overview

RecombDNA_sess2.jpg

This session will cover 1) what restriction enzymes are and how they cut DNA, 2) the different types of restriction enzymes and the advantages and disadvantages of using them, and 3) how restriction enzymes are used to create a recombinant DNA molecule.

Learning Objectives

  • To understand what a restriction endonuclease (restriction enzyme) is and how it functions.
  • To identify restriction enzyme(s) recognition sites given a section of DNA.
  • To understand the difference between enzymes that cut double-stranded DNA to produce a region of single-stranded DNA and those that do not, and the appropriate use of each type of enzyme.
  • To determine which restriction enzyme to use to create a desired piece of recombinant DNA.
  • To know the function of DNA ligase.
  • To understand what a vector is, what the minimum requirements are, and how it is used.

Session Activities

Lecture Video

Watch the lecture video excerpt

Check Yourself

 

 

Session Activities

Lecture Video

Watch the lecture video excerpt

Check Yourself

 

 

Session Activities

Lecture Video

Watch the lecture video excerpt

Check Yourself

You want to:

  1. Obtain the human insulin gene.
  2. Cut chosen vector and ligate the human insulin gene into vector.
  3. Use this ligation mixture to transform E. coli cells.
  4. Transfer E. coli cells to growth media.
  5. Select for E. coli cells that have obtained any vector.

Each of the steps listed above requires a specific DNA sequence found on the vector.

 

 

Session Activities

Practice Problems

Further Study

Suggested topics for further study in an introductory-level Biology textbook

  • Steps involved in a basic cloning strategy

Useful Links

 

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