Book contents
- Frontmatter
- Contents
- List of contributors
- Preface
- General advice
- 1 Observational Exercises on Parasites
- 2 Ecology
- 3 Physiology and Biochemistry
- 4 Pathology and Immunology
- 5 Chemotherapy
- 6 Molecular Parasitology
- 6.1 Purification of DNA
- 6.2 DNA digestion and gel electrophoresis
- 6.3 Restriction enzyme mapping
- 6.4 Construction of a genomic library
- 6.5 Detection and differentiation of Entamoeba histolytica and E. dispar by PCR
- 6.6 Differentiation between parasite species by agglutination and detection of parasite surface carbohydrates, using non-conjugated lectins
- 6.7 Tentative identification of parasite and tissue surface carbohydrates by conjugated lectins
- 7 Behaviour
- Appendix 1 Reagent index
- Appendix 2 UK suppliers
- Appendix 3 US suppliers
- Index
6.2 - DNA digestion and gel electrophoresis
Published online by Cambridge University Press: 05 June 2012
- Frontmatter
- Contents
- List of contributors
- Preface
- General advice
- 1 Observational Exercises on Parasites
- 2 Ecology
- 3 Physiology and Biochemistry
- 4 Pathology and Immunology
- 5 Chemotherapy
- 6 Molecular Parasitology
- 6.1 Purification of DNA
- 6.2 DNA digestion and gel electrophoresis
- 6.3 Restriction enzyme mapping
- 6.4 Construction of a genomic library
- 6.5 Detection and differentiation of Entamoeba histolytica and E. dispar by PCR
- 6.6 Differentiation between parasite species by agglutination and detection of parasite surface carbohydrates, using non-conjugated lectins
- 6.7 Tentative identification of parasite and tissue surface carbohydrates by conjugated lectins
- 7 Behaviour
- Appendix 1 Reagent index
- Appendix 2 UK suppliers
- Appendix 3 US suppliers
- Index
Summary
Aims and objectives
This exercise aims to perform spectrophotometric analysis of Escherichia coli DNA, followed by endonucleolytic digestion of this bacterial genome and DNA from a variety of other sources with the restriction enzyme, EcoRI.
The specific objectives of this practical are:
To measure the A260 and A280 of E. coli DNA.
To perform restriction digests with EcoRI.
To prepare an agarose gel.
To analyse the restriction digests by agarose gel electrophoresis.
Introduction
The yield and purity of isolated DNA can be estimated relatively easily using ultraviolet (UV) spectrophotometry. A further test of DNA purity is to assess the extent that it can be cut with restriction endonucleases, enzymes that recognise and make a double-stranded cut at specific nucleotide sequences. This is an essential technique used in the construction of gene libraries and the analysis of cloned genes. The digestion products are separated by size on an agarose gel and visualised under UV illumination, after staining with the fluorescent dye ethidium bromide.
Laboratory equipment and consumables
(per student or group)
Equipment
UV spectrophotometer and quartz cuvettes P20, P200 and P1000 Gilson pipetmen or equivalents 37°C waterbath; 65°C waterbath
Heater/stirrer, magnetic stirrer bar
Heat-resistant gloves
Agarose gel electrophoresis equipment and combs
Power supply for running gels
UV transilluminator and photographic system
Consumables
Latex gloves in a range of sizes
Sterile tips for pipetmen
Ice bucket and wet ice
EcoRI restriction enzyme (5 units/μ1)
Sterile distilled water and TE buffer
- Type
- Chapter
- Information
- Practical Exercises in Parasitology , pp. 343 - 348Publisher: Cambridge University PressPrint publication year: 2001