Agarose Gel

Electrophoresis of DNA
Mejico, Aia
Melchor, Kevin
Mendoza, Angelo
Mendoza, Edrian
Mendoza, Janel

Introduction

Gel Electrophoresis
Method for separation and analysis of
macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and
charge

As a general rule
Shorter molecules move faster and migrate
farther than longer ones because shorter
molecules migrate more easily through the
pores of the gel.
This phenomenon is called sieving.

Separation of DNA Fragments by Gel

Electrophoresis
Two Types
Polyacrylamide gels
Agarose gels

Polyacrylamide Gel
Separate DNA fragments up to 1000 bp long
Higher resolving power than agarose
Separate fragments that differ in length by
single nucleotide
Separating proteins ranging in size from 5 to
2,000 kDa due to the uniform pore size

Polyacrylamide Gel
Run in vertical direction
Used in DNA sequencing
Traditional DNA sequencing techniques such
as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA
fragments differing by a single base-pair in
length so the sequence could be read

Agarose Gel
Separate mixtures of fragments up to 20 kb
Easier to prepare
Lower resolution
Agarose gels do not have a uniform pore
size, but are optimal for electrophoresis of
proteins that are larger than 200 kDa

Agarose Gel
Entire chromosomes containing millions of
nucleotides can be separated on these gels
by applying pulsed electric fields in different
directions
Used in DNA separation

Objectives
To be able to separate the DNA using
Agarose Gel Electrophoresis
To be able to answer the guide questions for
this experiment

Materials

Materials
Agarose
Tris Acetate EDTA buffer
Fast Blast DNA
Tracking Dye

Materials
Horizontal Gel
Apparatus and combs
Power Supply
Gel Documentation
Apparatus
Ultra Rocker Rocking
Platform

Procedure

Procedure
A. Assembly of Agarose Gel Electrophoretic
Cell

Procedure
B. Agarose Gel Preparation
1% Agarose in 100 ml
TAE buffer

Solubilize, then cool to

50-60 C before pouring
in gel holder

Well-forming comb is
placed in one edge of
the gel

Combs were removed

gently

TAE buffer is poured

into gel tank completely
covering the gel

Gel is poured in the

holder/chamber. After
20 mins, it should be
hard, milky, and opaque

Procedure
C. Loading of DNA Samples
10 ul of PV92 XC loading dye was
added to each PCR tube except
MMR (Molecular Mass Ruler)

The samples were loaded in the 8

wells using a clean tip per sample

Procedure
La Sample
ne

Load
Volum
e

MMR (DNA
Standard)

10 ul

Homozygous (+/
+) control

10 ul

Homozygous
(+/-) control

10 ul

Heterozygous
(+/-) control

10 ul

Student 1

20 ul

Student 2

20 ul

Procedure
C. Loading of DNA Sample
Lid was secured on the gel
box, electrical leads connected
to the power supply

Turn on power supply, set to

100 V and electrophorese for
30 minutes

Turn off power after

electrophoresis and gently
nudge the gel into staining tray

Procedure
D. QUICK Staining of Agarose Gel in 100X Fast
Blast DNAStain
- For quick visualization of DNA bands in 15
minutes
- convenient, safe, and nontoxic alternative to
ethidium bromide

Procedure
Stain gels for 2-3
minutes

Rinse gels in warm tap

water for 10 seconds

Visualize DNA in the gel

documentation system

Wash gels in warm tap

water for 5 minutes twice

Examine the gels for

expected DNA bands.
Bands develop 5-15
minutes after 2nd wash

Results and Discussion

Results

Expected

Actual

Discussion

Discussion
detect Alu(300bp) sequence on
PV92(641bp) region in chromosome 16
Samples with Alu sequence will travel less
because of its bigger size

Sources of Errors

Low concentration of loaded sample

Prolonged time prior to visualization
Well contamination
Duration of electrophoresis
Improper loading technique
Gel problem

Guide Questions 4 and 5

What is the role of ethidium bromide and

what precautions would be observed when
handling this reagent? Why?
lEthidium bromide: intercalating agent
Used a nucleic acid stain
Fluoresce with a red-orange color
Aromatic
Tricyclic structure with aniline groups
on either side of pyridine
(a six- atom, nitrogen-containing,
aromatic ring)

Moves into the hydrophobic environment between the base

pairs
lAway from the water molecules (water: fluorescent quencher)
lRemoval of water: ethidium bromide will fluoresce

Strongly mutagenic
lIntercalates into double stranded DNA
lWear gloves (latex or nitrile rubber gloves)
Laboratory coat and goggles

When using a UV transilluminator,

what safety precaution should also be
observed and why?
lUsed in visualization of nucleic
acids stained with uorescent
dyes such as ethidium bromide
and acridine orange in gel
electrophoresis
lEmits a short wave UV light
lIt can cause severe damage
with very short exposure
periods

Exposure and Hazards of UV

lExposure to UV light: a serious threat to both the eye and
skin
lPhotokeratitis is an inflammation of the cornea (outer
protective coating of the eye) that is caused by exposure to
ultraviolet radiation
lEye injury can occur due to very brief exposure or with
just a flash of intense UV
lErythema is sunburn of the skin and can occur within a
few seconds of exposure to a concentrated form of UV.
lProlonged exposure to ultraviolet light also causes
premature aging and cancer of the skin

Laboratory Safety Precautions

Transilluminators are never to be used without the
protective shield in place
lNever operate without the safety hood or cover in place
when capturing gel images
lAlways wear protective equipment such as gloves, face
shields, and long sleeves lab coats
lUse thick gloves such as thick nitrile or latex gloves
Never allow the eyes or skin to be exposed to UV light