agarose gel electrophoresis

Agarose Gel Electrophoresis

Introduction

What is Gel Electrophoresis?

It is a basic biotechnology technique that separates macromolecules according to their size and charge

There are two types of gel electrophoresis namely Polyacrylamide gel and Agarose gel electrophoresis

What is its application?

It is frequently used to analyze and manipulate samples of DNA, RNA, or proteins.

Introduction

What to expect?

• Size and net charge are factors that together

determine how quickly molecules will travel

through the gel, and thus what their migration

distance will be.

– Small size vs. Large size

– Strong charge vs. weak charge

Introduction

• Short repetitive interspersed Elements (SINE)

– Consists of relatively short sequences (10 to a few

hundred base pairs in length)

– Exist at numerous places throughout the genome

– The Alu sequence

Introduction

• The Alu Sequence

– 300 base pair sequence that exist at more than

500,000 places throughout the human genome

– Function not completely understood

• Doesn’t code for any type of protein or DNA

• PV92 Locus on Chromosome 16

– homozygous or heterozygous

Objectives

• to separate and fractionate (using Agarose Gel

Electrophoresis) the isolated DNA in the

previous experiment

• to be able to draw conclusions based on

different sizes and charges that migrate

through a gel during electrophoresis

• to determine whether the subject is

homozygous or heterozygous for the alu gene

PROCEDURE

Assembly of Agarose Gel

Electrophoretic Cell

Agarose Gel Preparation

• 1% agarose in 100mL of 1x TAE buffer

• Solubilized in heat

• Cool down to 50 – 60 C

• Place the comb near the edge

• Pour gel over the chamber

• Pour 1x TAE buffer into the gel tank

• Remove combs

Loading of DNA samples

Lane Sample Load Volume

1 MMR (DNA standard) 10 ul

2 Homozygous (+/+) Control 10 ul

3 Homozygous (-/-) Control 10 ul

4 Heterozygous (+/-) Control 10 ul

5 Student 1 20 ul

6 Student 2 20 ul

7 Student 3 20 ul

8 Student 4 20 ul

Agarose Gel Electrophoresis

• DNA Separation

– Positive electric charge is applied to the negatively

charged nucleic acid molecules

– Shorter molecules move faster and migrate

farther than longer ones

– Migration affected by factors such as pore size,

voltage used and length of DNA sample

METHODS

D. Staining

1. Stain gels for 2-3 min with 100x Fast Blast

DNA Stain

METHODS

D. Staining

2. Rinse gels for 10 seconds with 500-700mL

of clean, warm (40-55°C) tap water.

METHODS

D. Staining

3. Wash with 500-700mL of clean, warm tap

water twice for 5min each.

METHODS

D. Staining

4. Examine for DNA bands (fuzzy at first)

METHODS

E. Visualization of DNA

Gel Documentation System

Results and Discussion

Alu

• Small, repetitive DNA elements of around 300 bp

repeated almost 500,000x throughout the human

genome

• Dimorphic – insertion may be present or absent on

each of the paired chromosomes of different people

• PV92 region of chromosome 16

– 641 bp intron

– 300 bp insertion (Alu)

Results

Lane Sample

1 MMR

2 (+/+) control

3 (-/-) control

4 (+/-) control

5 Student 1 (cheek)

6 Student 2 (cheek)

7 Student 3 (hair)

8 Student 4 (hair)

1 2 3 4 5 6 7 8

Lane Sample Base Pairs Genotype

1 DNA Standard

2Homozygous (+/+)

Control941

Homozygous (+/+)

3Homozygous (-/-)

Control641

Homozygous (-/-)

4Heterozygous (+/-)

Control941 and 641

Heterozygous (+/-)

5 Student 1 941 and 641 Heterozygous (+/-)

6 Student 2 941 Homozygous (+/+)

7 Student 3 941 Homozygous (+/+)

8 Student 4 941 and 641 Heterozygous (+/-)

Guide Questions

If ethidium bromide were to be used

as DNA visualizing agent, what

precautions would be observed

when handling this reagent? Why?

Ethidium Bromide

• 3,8-Diamino-5-ethyl-6-

phenylphenanthridinium

bromide

• intercalating agent

Ethidium Bromide

• Red cationic fluorescent

dye

• Visualize DNAs and

RNAs in electrophoresis

gels

• Fluoresces readily into

an orange/ reddish-

brown color when

exposed to UV light

Ethidium Bromide is a Mutagen

• May cause genetic damage

• Moderately toxic after an acute exposure

• Can be absorbed through the skin

• Irritant to skin, eyes, mouth and URT

Safety First!

• Substitute.

• Work in a suitable environment.

• Place in shatter-proof, leak proof container when transporting.

• Maintain a clean setting.

• Use PPEs.

• Emergency exposure procedures

• Dispose properly.

Personal Protective Equipments

• Protective clothing

– As much coverage as possible

• Eye protection

– Safety glasses with side shields

– Chemical splash goggles

• Gloves

– Disposable nitrile gloves, change frequently

– Handwash thoroughly after

Emergency Exposure Procedure

• IMMEDIATE medical attention

• Eyes – irrigate for 15 mins

• Skin – wash with soap and water

• Swallowed/inhaled – medical attention

Proper Disposal

• All contaminated articles must be treated as

hazardous waste and labeled as such.

When using a UV transilluminator to

visualize DNA what safety

precautions should be observed and

why?

UV Transilluminator

• Emit relatively high

levels of UV radiation

Effects of UV Radiation

• Photokeratitis

• Ocular cataracts

• Erythema

• Blistering

• Skin aging

• Skin cancer

Safety First!

• Substitute.

• PPEs (mask, gown, nitrile gloves)

• Shielding

• Warning signs