Aims and objectives

This exercise is designed to demonstrate the construction of a simple genomic DNA library. It can be adapted for use with any organism including parasites. The version given here is for the construction of a Plasmodium falciparum library.

The specific objectives of this exercise are:

1. To perform a ligation reaction.

2. To prepare competent E. coli cells.

3. To transform and plate these competent cells.

4. To grow small-scale cultures from recombinant colonies.

5. To prepare plasmid DNA from recombinants.

6. To analyse recombinant plasmids by agarose gel electrophoresis.

Introduction

The work to be described is aimed at the construction of a genomic library from an isolate of P. falciparum such as might be undertaken to analyse a specific gene. This type of cloning experiment is fundamental to a whole range of analyses in molecular biology.

Laboratory equipment and consumables

(per student or group)

Equipment

P20, P200 and P1000 Gilson pipetmen or equivalents

Waste beaker for chemically contaminated plasticware

Autoclavable waste bags for bacterially contaminated waste 37°C, 42°C and 65°C waterbaths

Bench-top centrifuge capable of producing 1000g 37°C incubator, static and orbital

Vortex machine

‘Camping gaz’ and lighter

Vacuum pump and desiccator

Eppendorf microfuge or equivalent

Heater/stirrer and magnetic stirring bar

Agarose gel electrophoresis apparatus and combs

Power supply for running gels

Photographic system for recording results

Safety glasses