age power point

Agarose Gel Electrophoresis of DNA

By:Justin Wilfred Tobias

Katherine Yap

Introduction

Electrophoresis an analytical process used to examine the

movement of charged molecules through a support medium under the influence of an electric field

Introduction

Equation 1:

E = electric field in volts/cm, q = net charge on the molecule, f = frictional coefficient which depends on the mass and shape of the molecule and v = velocity of the molecule

Eqv

f=

Introduction

• Under constant-voltage conditions, the movement of a charged molecule is dependent on the ratio of q/f.

• The f of molecules with similar conformation varies with size but not in shape.

• These conditions make equation 1 highly dependent on the charge (q) and mass dependence of f which means that molecules migrate in an electric field at a rate proportional to their charge-to-mass ratio.

Introduction

Equation 2:

v Eq q

E Ef fm= = =

Introduction• Equation 2 does not adequately describe the

electrophoretic process since important factors such as interaction of migrating molecules with the support medium and shielding of the molecules by the buffer ions are not accounted in the equation.

• This means that electrophoresis is not useful for describing specific details about the shape of a molecule but it is applied in the analysis of purity and size of macromolecules.

Introduction

Basis for the analysis and separation of electrophoretic methods:

each molecule in a mixture is expected to have a unique charge and size and hence, its mobility is also unique

Introduction

Uses of electrophoretic techniques:

analysis of amino acids, peptides, proteins, nucleotides, nucleic acids and other charged molecules

Types of Electrophoresis

Based on their support medium:1.Cellulose2.Gel

Cellulose

• used as a support medium for low-molecular weight biomolecules such as amino acids and carbohydrates

Gel

• widely used as support for larger molecules

• can either be polyacrylamide or agarose

• polyacrylamide - used for shorter fragments

• agarose - used for larger fragments

Gel Electrophoresis

• no void volume in the matrix• continuous network of pores throughout the

gel• comparable to a single bead in gel filtration• small molecules are eluted first before the

large ones• usually carried out at basic pH, where most

biological molecules are anionic hence they move toward the anode

• Two commonly used electrophoretic techniques for DNA analysis:

1.polyacrylamide gel electrophoresis (PAGE)2.agarose gel electrophoresis (AGE)

PAGE AGE

size range, bp 6 – 1000 70 – 80 000

concentrations 20% - 3% acrylamide 3% - 0.01% agarose

PAGE

• prepared by the free radical polymerization of acrylamide and the cross-linking agent N,N’-methylene-bis-acrylamide

• enhanced resolution of sample components because the separation is based on both molecular sieving and electrophoretic mobility

• standard gel for protein separation is 7.5% polyacrylamide = molecular size range of 10,000 to 1,000,000 daltons (best resolution is in the range of 30,000 to 300,000 daltons)

PAGE

• resolving power and molecular size range of a gel depend on the concentrations of acrylamide and bis-acrylamide

• M = gels have larger pores which allows analysis of higher-molecular-weight biomolecules

• M = gels with smaller pores which allow analysis of lower-molecular-weight biomolecules

PAGE

Advantages of acrylamide polymerization in electrophoresis:

• high resolving power for small and moderately sized proteins and nucleic acids (up to approximately 1x106 daltons)

• acceptance of relatively large sample sizes • minimal interactions of the migrating molecules

with the matrix • physical stability of the matrix

AGE

• used to characterize RNA and DNA in the range of 200 to 50,000 base pairs (50 kilobase)

• agarose concentrations of 0.3 to 2.0% are the most effective for nucleic acid separation

• gels with less than 0.5% agarose are rather fragile and must be used in a horizontal configuration

Agarose

• a linear polymer of galactopyranose derivatives

• a natural polysaccharide that is extracted and purified from seaweed

Properties of Agarose

• form inert matrix• high sulfate content• high gel strength • specific gel point

Structure of Agarose

PAGE vs AGE

Parameter PAGE AGESupport medium Polyacrylamide Agarose

Mode of gelling Polymerization Non-covalent interactions

Gel preparation More difficult Easier

Visualization reagent Coomassie brilliant blue Ethidium bromide

Migration anode anode

Separation range Small fragments Moderate to larged size

Resolving power Higher Lower

DNA purity Higher Lower

EEO

• acronym for Electroendosmosis• drift of a fluid through an aqueous gel towards

an electrode during electrophoresis• drift occurs when electrically neutral, or nearly

neutral, molecules are present in a sample to be electrophoresed and the gel medium carries a charge

EEO• when agarose is the medium, anionic residues

such as ester sulfate and pyruvate groups are present and impart a net negative charge to the gel

• although the gel itself can't move anodally, the water sphere around it is pulled or distorted toward the cathode by hydrated cations associated with the bound anions

• as a result, neutral molecules in the sample are gradually pulled towards the cathode with the water

EEO

• results in blurred zones and drying of the gel in the anodal area of flatbed gels

• normally seen as a negative effect, yet a few methods take advantage of this effect to achieve separation or detection results (MEKC in capillary electrophoresis and counter immunoelectrophoresis)

Preparation of gels:

• High quality low-melting agarose (which melts at 65°C and sets at 30°C)

• low EEO DNA molecules remain double stranded

Experiment

OBJECTIVES• To characterize the extracted DNA (maggot,

cockroach, liver) using AGE• To describe the resulting band profile• To identify the factors affecting the quality of

the resulting band profile

Methodology

• Buffer Preparation• Gel Preparation• Sample Preparation and Loading• Running the Gel• Viewing the Gel

Buffer Preparation50x TAE 10x Tris-phosphate 10x TBE

Tris base – 242 g Tris base – 108 g Tris base – 108 g

Glacial HOAc – 57.1 mL 85% H3PO4 – 15.5 mL H3BO3 – 55 g

0.5 M EDTA (pH 8) – 100 mL 0.5 M EDTA (pH 8) – 40 mL Na2EDTA.2H2O – 9.3 g

Diluted to 1 L Diluted to 1 L Diluted to 1 mL

TAE Working Solution Tris-phosphate Working Solution

TBE Working Solution

Tris acetate – 0.04 M Tris-phosphate – 0.08 M Tris-borate (pH 8.3) – 0.089 M

EDTA – 0.001 M EDTA – 0.002 M Disodium EDTA – 0.025 M

Buffers

• Tris-Acetate-EDTA (TAE)• Tris-Borate-EDTA (TBE) • Tris-phosphate

Tris-phosphate

• good buffering capacity • but phosphate will co-precipitate if DNA is

ethanol precipitated

TBE

• better resolution than TAE • but it interacts with the nucleic acids resulting

to a lower recovery of DNA in gels• more expensive than TAE

TAE

• faster running time compared to TBE • can be used for preparative work• low buffering capacity and cannot be used for

long running times

TAE

Composed of:• Tris-base • glacial acetic acid• EDTA

TAE

• Glacial acetic acid increases the ionic strength of the solution.

• EDTA is used as a chelating agent for the divalent cations that promote the intermolecular interaction of the DNA with denaturing enzymes like nucleases and restriction enzymes.

• TAE is the buffer used in this experiment because linear double stranded DNA tends to run faster in it.

Gel Preparation

• weighed 0.25 g gel powder and dissolved in 12.5 mL of 1x TAE buffer

• agarose mixture was heated and was strirred occasionally

• mixture was not boiled and the resulting solution was homogenous, transparent and fluid

Gel Preparation

• gel was allowed to cool since hot gels (T > 60°C) could deform the gel tray (+ 150 µL EtBr)

• gel was poured carefully so as not to introduce air bubbles in the mold

• comb was placed in the gel and in one swift fluid motion the comb was removed after the gel has solidified

• wells were then flushed with buffer before loading the sample

• 1.0% agarose gel was prepared because it coincides with the range from 0.3-2.0% of which the nucleic acid separation is more effective

Gel Structure of Agarose

Sample Preparation and Loading

Loading buffer preparation:• 30% Ficoll solution + marker dyes (0.25%

bromphenol blue or 0.25% cyanole) + 0.2 M EDTA (pH 8)

• dissolved in 10 volumes of buffer

Sample Preparation and Loading

30% Ficoll solution• increases density of the solution• preferred over glycerol and sucrose because it

minimizes tailing at the edges0.25% bromphenol/0.25% cyanole• used as tracking dyesEDTA• disables restriction enzymes and nucleases

during the run

Sample Preparation and Loading

• 60 µL loading buffer + 140 µL DNA sample • draw the “spot” up and down using a pipette

tip to mix• load 20 µL of the mixture on the well

Running the Gel

• Fill the gel chamber with running buffer.• Set and maintain the power supply to 100V.• Turn the apparatus off after 30 – 35 minutes.

Viewing the Gel

• gel was removed from the set-up and was placed in a transparent flat bottomed container

• 0.5-1 drop of ethidium bromide solution was then added over the surface of the gel

• (the addition of ethidium bromide is done in this part if in-gel staining is not performed)

• the gel was then immediately viewed under a UV light

Viewing the Gel

• bands were seen as orange under UV light• The visualization and picture taking of the gel

should be performed shortly after running the gel because DNA will diffuse over time and the bands will become blurry.

EtBr

• a fluorescent dye that intercalates or inserts between the bases of a double-stranded DNA

• forms an EtBr-nucleic acid complex with the sample which exhibits a more intense fluorescence than a free EtBr in the UV region

• absorbs at 260 nm to 300 nm and has a maximum emission at 540 nm

In-gel Staining

Advantages:• uses fewer glassware • UV light can be used to monitor the runDisadvantage:• the binding of ethidium bromide alters a

DNA’s mass and rigidity, and therefore its mobility

Conformations

• supercoiled • linear • nicked circles

Conformations

• The supercoiled conformation is small, compact and usually has the greatest mobility followed by the rodlike, linear form of molecules.

• The nicked circles or extended circular form molecules migrate more slowly.

• The relative electrophoretic mobility of the three forms depends on experimental conditions such as agarose concentration and ionic strength.

Conformations

• The addition of ethidium bromide affects the electrophoretic mobility of the three conformations.

• If ethidium bromide is added to the supercoiled form, the dye intercalates between the stacked DNA bases causing unwinding of some of the negative supercoils.

• As the concentration of ethidium bromide is increased, more and more of the negative supercoils are removed until no more are present in the DNA.

• The conformational change of the DNA supercoil can be monitored by electrophoresis because the mobility decreases with each unwinding step.

• The linear and nicked circles forms, under the same conditions of increasing ethidium bromide concentration, will also show a gradual decrease in electrophoretic mobility.

AGE Set-up

Components of AGE Set-up

• power supply - introduces an applied electric field in the gel system

• electrodes - where the separation of charge occurs

• electrophoresis chamber - where the melted agarose assumes it shape when it solidifies

• sample combs - where the melted agarose is poured to form sample wells in the gel

Components of AGE Set-up

• running buffer - provides the ions in the electrophoretic system to support conductivity

• loading buffer - migrates in the gel and allows visual monitoring or the extent of electrophoresis

• ethidium bromide - a fluorescent dye used for staining nucleic acids

• transilluminators - used to visualize ethidium bromide-stained DNA in gels

…

• The separation of molecules in AGE is achieved by moving negatively charged nucleic acid molecules through an agarose matrix under the influence of an electric field.

• The negative charge of DNA in neutral pH is due to the presence of its negatively charged phosphate backbone.

…

• Nucleic acids migrate in an agarose matrix at a rate that is inversely proportional to their size.

• A linear relationship exists between mobility and the logarithm of kilobase pairs (or molecular weight) of a DNA fragment.

• A standard curve may be prepared by including on the gel a sample containing DNA fragments of known molecular weights.

Factors Affecting AGE

• agarose concentration • voltage • running buffer • ethidium bromide

Agarose Concentration

• Improper agarose concentration will lead to poorly resolved bands.

• High agarose concentration is used in the separation of smaller DNA.

• Low agarose concentration is for the separation of larger DNA.

Voltage

• As voltage is increased, larger fragments migrate faster than small fragments.

• This is because DNA becomes rigid under high voltage condition and so, DNA travels faster.

• Low voltage means better resolution but longer running time.

Running Buffer

• provides ions to support conductivity • absence of buffer would cause the DNA

samples not to migrate toward the anode • the criteria in choosing the appropriate buffer

system is the length of running time and if AGE will be used for the sample preparation

EtBr

• The binding of ethidium bromide to DNA will change the charge, weight, conformation and flexibility of the DNA molecule.

• The mobility of DNA decreases as ethidium bromide binds to it due to stiffening.

Errors…

• reagents used• sample preparation