Aims and objectives

This exercise is designed to demonstrate the isolation of genomic DNA; it can, in principle, be adapted for use with any organism, including parasites. Specifically, the objectives are:

1. To perform bacterial cell lysis.

2. To extract the cell lysate with phenol/chloroform.

3. To isolate the DNA by ethanol precipitation.

Introduction

DNA encodes genetic information for all known living organisms. A basic method in molecular biology is the isolation of DNA. The version given here is for the preparation of DNA from Escherichia coli. In this context the practical is useful for demonstrating some basic methods in molecular biology as well as introducing students to the use of bacteria as hosts for gene cloning. This is a prerequisite for more complex molecular biological methods including the isolation of parasite genes and analysis of their expression products.

Laboratory equipment and consumables

(per student or group)

Equipment

Safety glasses

P20, P200, P1000 Gilson pipetmen or equivalents

Waste beaker for chemically contaminated plasticware

Autoclavable waste bags for bacterially contaminated waste

Waste container for bacterial culture supernatant

Bench top centrifuge capable of producing 1000 g

Water bath (37 °C) with suitable racks for Universal tubes

Centrifuge capable of producing 8000g at 10 °C

Use of cold room/refrigerator and freezer

Consumables

Latex gloves in a range of sizes

Sterile tips for pipetmen

Universal tube containing a 25 ml culture of E.coli grown overnight at 37 °C in L-broth

3 ml of bacterial cell lysis buffer

300 μ1 of 10% (w/v) sodium dodecyl sulphate (SDS) in a microfuge tube