Biology Reference
In-Depth Information
CHAPTER 5
Some Basic Tools:
How to Cut, Paste, Copy, Measure,
Visualize, and Clone DNA
Chapter Outline
5.1 Overview 183
5.2 Introduction to a Basic Molecular Biology Experiment
184
5.2.1 The Pros and Cons of Kits 185
5.2.2 A Simple Cloning Experiment 186
5.3 Extracting DNA from Insects 187
5.3.1 DNA Extraction Resulting in Loss of the Specimens 187
5.3.2 DNA Extraction That Does Not Require Destroying the Specimens
188
5.3.3 Assessing the Quality of Extracted DNA
189
5.4 Precipitating Nucleic Acids
190
5.5 Shearing DNA 192
5.6 Cutting DNA with Restriction Endonucleases
192
5.7 Joining DNA Molecules 195
5.8 Growth, Maintenance, and Storage of
E. coli
196
5.9 Plasmids for Cloning in
E. coli
196
5.10 Transforming
E. coli
with Plasmids 201
5.11 Purifying Plasmid DNA from
E. coli
202
5.12 Electrophoresis in Agarose or Acrylamide Gels 204
5.13 Detecting, Viewing, and Photographing Nucleic Acids in Gels
206
5.14 Identifying Specific DNA by Southern Blot Analysis
206
5.15 Labeling DNA or RNA Probes 209
5.16 Removing DNA from Agarose Gels after Electrophoresis
210
5.17 Restriction-Site Mapping
211
General References
212
References Cited
213
5.1 Overview
Genetic engineers use several techniques to isolate DNA, cut and join mol-
ecules, and monitor the results. To clone a gene, determine its sequence, or
alter the genetic makeup of an arthropod, microbiological techniques are used,
