Restriction Endonucleases

Introduction

A bacteriophage is a virus that infects bacteria. A type of bacteriophage is bacteriophage lambda. This specific bacteriophage infects E. Coli. It is composed of protein and double stranded DNA ЎVdsDNA). The DNA of the phage contains around 50,000 base pairs and codes for 50 proteins. At both ends of the DNA of the phage, there are cohesive ends, which are composed of 12 nucleotides. Both ends compliment each other, which makes the DNA circular once together. This circular DNA is usually present in an infected bacterial cell. This protects the DNA to be degraded by the cell.

In this laboratory experiment, restriction enzymes were used to analyze the DNA of the lambda phage. Restriction enzymes cut DNA in specific locations of the DNA sequence. The restriction enzymes used were EcoR1 and Bam H1. Both of these restriction enzymes cut DNA into six fragments.

Materials

Agarose

Gel Electrophoresis Apparatus

Gel Comb

Gel Mold

100x Triacetate EDTA Buffer with a pH of 8.0

Methylene Blue Stain

Microwave

Micropipette

Ice Bath

Water Bath 37ўXC (Incubator)

Water Bath 70ўXC (Incubator)

Phage Lambda DNA (uncut)

EcoR1- Restriction Enzyme

EcoR1 + Bam H1 ЎV Mixture of Restriction Enzymes

Distilled Water

Methods

Ўч Preparing the DNA samples

In this lab experiment, four small tubes were labeled 1-4. Once labeled, 10Јgl of the following material was placed in each of the tubes as shown in Table 1.1:

Table 1.1 Sample Placement in small Tubes

TUBE # Name of Sample

1 Distilled Water

2 Eco-R1 Buffer solution

3 EcoR1 + Bam H1 Mixture

4 EcoR1 + Bam H1 Mixture

After each of the solutions were put