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:

1. To measure the A260 and A280 of E. coli DNA.

2. To perform restriction digests with EcoRI.

3. To prepare an agarose gel.

4. 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