Electrophoresis Modified

7/31/2019 Electrophoresis Modified

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Basics

Speed dependent on their charge, shape,and size, hence electrophoresis has beenextensively developed for molecular

separations.

It is used chiefly for analysis and

purification of very large molecules suchas proteins and nucleic acids,

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Factors AffectingElectrophoresis

1. The Electric Field.

1. Voltage.

b. Current.

c. Resistance.

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2. The Sample

a. Charge.

b. Shape.

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3. The Buffer

a. Composition.

b. Concentration.

c. pH.

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4. The Supporting Medium.

a. Adsorption.

b. Electro – osmosis.

c. Molecular Sieving.

Gel Electrophoresis.

Separation of high molecular weightsubstances such as proteins andnucleic acids

Improved resolution since the gel iswater insoluble and hydrophilic.

Gel Electrophoresis.

Eg: Starch, agar and polyacrylamide.

Molecular sieving property of gels helpsto separate large ionic compoundshaving similar charge but different sizeand shape.

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Starch Gel Electrophoresis

Starch gels are prepared by heatingand cooling mixture of partiallyhydrolysed starch resulting in a semi

rigid gel formation. Good for separating complex mixtures

of structural molecules andphysiologically active proteins.

Analysis of isoenzyme patterns(zymograms)

Agarose Gel

Electrophoresis Agar is a cheap,

non toxic,

chemically illdefined,complex,powdery mixture.

Contains twogalactose basedpolymers,agarose and

agaropectin.

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Agarose GelElectrophoresis

Dissolves in boiling aqueous buffers andforms a gel at 38°C.

Large pore size and low frictional resistance.

Facilitates separation of macromolecules. Suitable for chemical staining after

separation.

Excellent for detection of antigenic proteinsby isotachophoresis andimmunoelectrophoresis.

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DNA separation usingAgarose Gel Electrophoresis

Gel electrophoresis is a widely usedtechnique for the analysis of nucleic acidsand proteins. Agarose gel electrophoresisis routinely used for the preparation andanalysis of DNA.

Equipment

1. A power pack.

Supplies direct

current betweenthe electrodesbetween theelectrophoresis

Equipment

2. ElectrophoresisUnit

a. Electrodes.

b. Buffer reservoirs.

c. Support medium.

d. Transparentinsulating cover

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• DNA is negatively charged.

• When placed in an electrical field, DNA will migrate toward thepositivepole (anode).

• An agarose gel is used to slow the movement of DNA andseparate by size.

Scanning ElectronMicrograph of Agarose Gel

• Polymerized agarose isporous,

allowing for the

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How fast will the DNAmigrate?strength of the electrical field, buffer, density of agarose

gel…Size of the DNA!

*Small DNA move faster than large DNA…gel electrophoresis separates DNA according tosize

smalllarge

Within an agarose gel, linear DNA migrate

inversely proportional to the log10 of theirmolecular weight.

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Making an AgaroseGel

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An agarose gel is

prepared by combiningagarose powder and abuffer solution.

Agarose

Buffer

Flask for boiling

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Casting tray

Power supply

Gel tank Cover

Electrical leads

ElectrophoresisEquipment

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Gel casting tray &

Preparing the Casting

7/3/12 Seal the edges of the casting tray and put in the combs. Place thecasting tray on a level surface. None of the gel combs should betouching the surface of the casting tray.

Preparing the Casting Tray

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Agarose

Buffer Solution

Combine the agarose powder and buffer solution. Use a flaskthat is several times larger than the volume of buffer.

Melting the

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Agarose is insoluble at room temperature

(left). The agarose solution is boiled until clear

(right).Gently swirl the solution periodically when heating toallow all the grains of agarose to dissolve.

***Be careful when boiling - the agarose solution maybecome su erheated and ma boil violentl if it has

Melting theAgarose

Pouring the

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Allow the agarose solution to cool slightly (~60ºC) and thencarefully pour the melted agarose solution into the casting

tray. Avoid air bubbles.

Pouring thegel

7/3/12 Each of the gel combs should be submerged in the melted agarose

solution.

7/3/12 When cooled, the agarose polymerizes, forming a flexible gel.It should appear lighter in color when completely cooled (30-45

minutes). Carefully remove the combs and tape.

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Place the gel in the electrophoresischamber.

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buffer

Add enough electrophoresis buffer to cover the gel to adepth of at least 1 mm. Make sure each well is filled withbuffer.

Cathode(negative)

Anode(positive

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6X Loading Buffer:Bromophenol Blue (for

color)

Glycerol (for weight)

SamplePreparation

Mix the samples of DNA with the 6X sample loading buffer (w/tracking dye). This allows the samples to be seen when loadingonto the gel, and increases the density of the samples, causingthem to sink into the gel wells.

L di th

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Loading theGel

Carefully place the pipette tip over a well and gently expel thesample. The sample should sink into the well. Be careful not topuncture the gel with the pipette tip.

R i th

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Place the cover on the electrophoresis chamber, connecting theelectrical leads. Connect the electrical leads to the power supply.Be sure the leads are attached correctly - DNA migrates towardthe anode (red). When the power is turned on, bubbles should

form on the electrodes in the electrophoresis chamber.

Running theGel

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wells Bromophenol Blue

Cathode(-)

After the current is applied, make sure the Gel is running in thecorrect direction. Bromophenol blue will run in the samedirection as the DNA.

DNA(-)

DNA Ladder

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12,000bp

DNA LadderStandard

Inclusion of a DNA ladder (DNAs of know sizes) on thegel makes it easy to determine the sizes of unknownDNAs.

DNAmigratio

bromophenolblue

Note:bromophenolblue migratesat

approximatelythe same rateas a 300 bpDNA molecule

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As an alternative to purchasing costly DNA ladders, one can becreated using meal worm (Tenebrio molitor ) DNA and a restrictionenzyme.

http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf

Staining the

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Staining theGel

***CAUTION! Ethidium bromide is a powerful mutagen

and is moderately toxic. Gloves should be worn at

• Ethidium bromide binds to DNA and fluoresces under UVlight, allowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel and/or runningbuffer before the gel is run or the gel can be stained after ithas run.

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Safer alternatives to EthidiumBromide

Methylene Blue

BioRAD - Bio-Safe DNA Stain

• Ward’s - QUIKView DNA Stain

• Carolina BLU Stain

…othersadvantagesInexpensiveLess toxicNo UV light requiredNo hazardous wastedisposal

disadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time

St i i th

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Staining theGel

• Place the gel in the staining tray containing warm dilutedstain.• Allow the gel to stain for 25-30 minutes.• To remove excess stain, allow the gel to destain in water.

• Replace water several times for efficient destain.

Ethidi B id i lt i l t li ht t

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Ethidium Bromide requires an ultraviolet light source tovisualize

Visualizing the DNA (ethidium

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Visualizing the DNA (ethidiumbromide)

ß 5,000bp

DNA ladderDNA ladder

Product

1 2 3 4 5 6 7 8

+ - - + - + + -

Samples # 1, 4, 6 & 7 were positive for WolbachiaDNA

Primerdimers

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Visualizing the DNA (QuikVIEW stain)

2,000bp

DNA ladder

PCRProdu

+ - - - - + + - - + -

March 12, 20

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia

l l id l

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Polyacrylamide GelElectrophoresis.

Polyacrylamide gels are physicallytougher than agarose gels.

The gel forms when a mixed solution of

acrylamide and cross-linker monomersco-polymerize into long chains that arecovalently cross-linked.

The most common cross-linker is N,N'-methylenebisacrylamide (“bis”).

P l l id G l

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Porosity of gel is determined by relativeproportion of acrylamide monomer tocross linking agent

Gels may be defined in terms of total %of acrylamide present.

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SDS PAGE

Sodium dodecylsulphate polyacrylamidegel electrophoresis.

Principle: SDS is an anionic detergent

which binds to proteins causing theirdenaturation.

In excess of SDS the proteins have aconstant negative charge.

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SDS PAGE

Protein-SDS complex moves towardsanode.

Mobility inversely proportional to log of

molecular weight. Molecular weights of unknown proteins

can be determined.

P l l id G l

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Polyacrylamide Gel

Electrophoresis.

Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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y yElectrophoresis.

Polyacrylamide Gel

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y yElectrophoresis.

Polyacrylamide Gel

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y yElectrophoresis.

Polyacrylamide Gel

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y yElectrophoresis.

Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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Polyacrylamide Gel

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