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L6663 Sigma

Low-Cost Approach to PCR: Appropriate Transfer of Biomolecular Techniques

  •  ISBN-10 0-19-511926-6

  •  ISBN-13 978-0-19-511926-8

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Related Categories Books and Software, Labware, Molecular Biology Books, PCR Books
publication info   E. Harris, Oxford University Press, 1998, 328 pp., soft cover
mfr. no.   Oxford University Press

Description

General description

Despite the large impact of PCR, many applications remain within the confines of research and academic environment. Dr. Harris makes this technique accessible to researchers, physicians, and laboratory workers throughout the world. It provides practical details of the method, and technology transfer program that she developed. It serves as a guide for those with limited budgets, such as potential users in developing countries, those wishing to work abroad or in undergraduate labs. Specific applications are immediately useful to study of infectious diseases.

Table of Contents

1 Introduction
2 PCR Technology
2.1. Description of the Technique
2.1.1. Historical Overview
2.1.2. The Molecular Basis of PCR
2.1.3. Advantages and Disadvantages
2.2. Technical Details
2.2.1. Components of the PCR Mixture
2.2.2. Thermal Cycling Parameters
2.2.3. Optimizing
2.2.4. Potential Problems
2.2.5. Detection of Products
2.3. Frequently-Used PCR-Based Techniques
3 Principles of Sustainable Technology Transfer
3.1. A Low-Cost Methodology
3.1.1. Appropriate Technology
3.1.2. Alternative Techniques
3.1.3. Simplification of Protocols
3.1.4. In-House Preparation of Reagents
3.1.5. Recycling
3.1.6. Donated Materials
3.2. Knowledge-Based Participatory Transfer Process
3.3. Appropriate Application
3.3.1. General Considerations
3.3.2. An Evaluation Framework
3.3.3. Assessment Criteria for PCR
3.3.4. Case-by-Case Evaluation of PCR Applied to Infectious Diseases
3.4. Intra-Reginal Cooperation
4 Case Study: The AMB/ATT Program
4.1. Introduction
4.2. Program Description
4.2.1. Objectives
4.2.2. Format
4.3. Program Structure
4.3.1. Phase I
4.3.2. Phase II
4.3.3. Phase III
4.4. Program Development
4.5. Examples of Projects
4.6. International Courses
II Selected Protocols
5.1. PCR Protocols
5.1.1. Overview
5.1.2. Equipment, Materials, Control, and Procedures Common to All PCR Protocols
5.1.3. Dengue Virus
5.1.4. New World Leishmania
5.1.5. Mycobactrium tuberculosis
5.1.6. Plasmodium falciparum and Plasmodium vivax
5.1.7. Vibrio cholerae
5.1.8. Diarrheagenic E. coli and Shigella
5.1.9. Chlamydia trachomatis and Neisseria gonorrhoeae
5.1.10. Leptospira
5.1.11. Trypanosoma cruzi
5.2. Nonradioactive DNA Probes: V. cholerae Colony Blot
5.2.1. Labeling the Probe Using PCR
5.2.2. Preparation of the Colony Blot
5.2.3. Hybridization
5.2.4. Visualization
6 Rapid Cloning of PCR Products
6.1. Primer Design
6.2. Preparation of PCR Products for Cloning
6.2.1. Digestion of Vector and PCR Products
6.2.2. Preparative Agarose Gel
6.2.3. Purification of the Excised DNA Fragments Using Silica Particles
6.3. Ligation
6.3.1. Checking DNA Fragment Concentration by Agarose Gel Electrophoresis
6.3.2. Ligation Reaction
6.4. Preparation of Competent Cells and Transformation
6.4.1. Preparation of Competent Cells
6.4.2. Transformation
6.5. Checking Clones by PCR
6.6. Plasmid Purification
6.7. Analysis of Clones by Restriction Enzyme Digestion
6.8. Agarose Gel Electrophoresis
III Appendix
A. Construction of Laboratory Equipment
B. In-House Preparation of Reagents
B.1 Useful Formulas
B.2 Solutions
B.3 Preparation of Selected Reagents
B.4 DNA Size Markers
C. Inventory for a PCR Laboratory
D. Good Laboratory Practice
D.1 General Tips
D.2 Calibration of Adjustable Pipettors
E. Prevention of Cross-Contamination
F. PCR Troubleshooting Guide and Flow-Chart
G. Workshop Organization and Teaching Tips
H. Sample Charts and Worksheets
I. Useful World Wide Web Sites

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