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 ÐŽVdsDNA). 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 37ÑžXC (Incubator)

Water Bath 70ÑžXC (Incubator)

Phage Lambda DNA (uncut)

EcoR1- Restriction Enzyme

EcoR1 + Bam H1 ÐŽV 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 into the tubes, 5Јgl of lambda phage DNA was placed into each tube. In order to mix the solutions inside, the tubes were gently tapped. The tubes were then placed into the Water Bath 37ўXC Incubator for 50 minutes.

Ўч Preparing the Gel Electrophoresis

While the tubes are incubating, the gel electrophoresis apparatus was being set up. The concentration of the agarose gel needed is 1.2%. The number of grams needed to make 1.2% of agarose gel in 30mLs of Triacetate EDTA Buffer with a pH of 8.0 was calculated. It was calculated that 0.36 grams of the agarose powder needed to be used. Using the balance, 0.36 grams of agarose powder was weighed and then 30 mL of Triacetate EDTA Buffer with a pH of 8.0 was added to the agarose powder. The mixture of the buffer and powder were then heated in the microwave until the solution was clear (60 seconds). Once the solution was clear, it was then placed into the gel electrophoresis apparatus with the gel comb. The gel was left alone to solidify. When the gel was solid, the gel comb was removed leaving behind the wells.

Ўч Cont. of Preparation of DNA Samples

Once the gel is prepared and the incubation period reached 50 minutes, 5Јgl of gel electrophoresis sample buffer (Triacetate EDTA Buffer with a pH of 8.0), was placed into each of the four tubes. Tube #4 was then placed was transferred to the Water Bath 70ўXC (Incubator) for five minutes. When the five minutes have passed, the tube was then placed in the ice bath for a couple of minutes.

Ўч Gel Electrophoresis

15Ñ"Эl of each sample were loaded to the well using a micropipette. Samples and wells are listed below in Table 1.2.

Table 1.2 Sample Placement in Wells

WELL # Name of Sample

1 Tube #1

2 Tube #2

3 Tube #3

4 Tube #4

After the wells were loaded with the samples, the wells were sealed with agarose gel. This prevents the samples from overflowing in the gel. Once this was completed the gel was moved so that the wells were on the negative electrode side. Then 300mL of Triacetate EDTA Buffer with a pH of 8.0 was then added to the electrophoresis apparatus. The wires for the electrophoresis apparatus were then connected to the voltage base; one wire on the negative and the other on the positive. Then cover the gel with the lid and turning on the base to 120 mV. The electrophoresis was run for 20-30 minutes until the samples migrated around 1.5-2 mm of the positive electrode. This is observed by observing the bromophenol blue in the samples migrate 1.5-2mm of the positive electrode. Once the migration was complete, the gel was taken out of the apparatus and then placed in Methylene Blue dye overnight. The next day it was rinsed several times with distilled water and the distance of the bands were measured under the light.

Results

Table 1.3 Well #1 Distilled Water + Lambda Phage DNA

Cut