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Introduction to Recombinant DNA Technology v2.0
Source PCCAL, University of Bath
ISBN: 1 84211 013 6
Versions Available Internet
Programmer Mandy Gilbert
Subject Specialist Dr.Steve Moss
Summary
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Contents
1. Gene Cloning - an Overview 3. Manipulation of DNA/RNA
Introduction
  • Nucleases
  • Polymerases
  • Modifying Enzymes
  • Topoismerases
  • Ligases and Ligation
2. Purification of DNA/RNA 4. Manipulation of DNA - Restriction Enzymes
  • Purification of Total Cell DNA
  • Purification of Plasmid DNA - Alkaline Denaturation
  • EtBr-CsCI Density Gradient Centrifugation
  • Purification of Plasmid DNA - Other Methods
  • Plasmid Amplification
  • Purification of Total RNA
  • Purification of mRNA
  • Gel Analyses
  • Types of Restriction Enzymes
  • Finding the Results of Restriction Enzyme Use
  • Restriction Enzyme Mapping - Workshop
Summary of Section
Gene Cloning - an Overview
The student is given an overview of what gene cloning is, general techniques used in gene cloning, and applications of gene cloning within pharmacy and pharmacology
Purification of DNA/RNA
In this section, the student examines the experimental techniques used to purify the following types of DNA and RNA: total cell DNA, plasmid DNA (e.g. by alkaline denaturation, by EtBr-CsCl density gradient centrifugation), total RNA, and mRNA. The section also covers plasmid amplification.
Manipulation of DNA
The student is introduced to the range of enzymes used in gene cloning. The enzymes covered are nucleases, polymerases, modifying enzymes (alkaline phosphatase, polynucleotide kinase, terminal deoxynucleotidyl transferase), topoisomerases and ligases (including their use in the production of blunt and sticky ends).
Restriction Enzymes
The final section looks at the types of restriction enzymes and then goes on to discuss how restriction enzymes are used. The student can then attempt a workshop in which the student is asked to find and interpret results from restriction enzyme digestion using various restriction enzymes. The student's aim is to find out where the restriction enzyme cutting sites are on a selection of example DNA pieces.