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Agarose Gel Electrophoresis1PurposesTo understand the principle of Gel electrophoresisTo become familiar with the part of the electrophoresis setup2Merupakan teknik untuk memisahkan molekul berbasis muatan dan juga ukuran What is Electrophoresis?3Charged molecules are separated based on their electrical charge and size. Separation of a Mixture of Charged MoleculesCharge SeparationSize SeparationAnalyze

Identify

PurifyMixture of Charged MoleculesPositive MoleculesNegative Molecules4How Separation Occurs1- Electrical Charge:

Berdasarkan strukturnya, banyak molekul memiliki muatan baik itu negatif maupun positif, seperti DNA, RNA, Protein Molecules negative (anions) akan bergerak ke arah positive (anode).Molecules positive (cations) akan bergerak ke arah negatively (cathode).

52- Molecule Size:Material berpori dibuat dari mikropartikel yang nantinya akan berbentuk gel Partikel tersebut akan berperan sebagai saringan sehingga dapat memisahkan molekul berdasarkan ukurannya.Molekul yang kecil dapat bergerak lebih cepat dibanding molekul yang besar.

Porous MaterialProteins Entering Porous MaterialSmallest Move Fastest

How Separation Occurs6Size to molecular ratio Agarose karbohidrat komplek dari rumput laut Digunakan juga di industri pangan (ice cream, and jellies) dan banyak medium biologi.Ukuran porinya cukup besar sehingga dapat dengan cepat memisahkan molekul Polyacrylamide dari molekul akrilamidaKadang-kadang juga digunakan sebagai bahan pembentuk plastik Dibanding agarose, ukuran porinya lebih kecil jadi bagus untuk memisahkan molekul yang kecil

Acrylic AcidGels dapat dibentuk dari agarose maupun akrilamida

Red Sea WeedGel Electrophoresis7Agarose GelMaterial perpori dari rumput lautBerperan sebagai penyaring untuk memisahkan molecules.Konsentrasi yang digunakan akan mempengaruhi ukuran poriLow conc. = larger pores better resolution of larger DNA fragmentsHigh conc. = smaller pores better resolution of smaller DNA fragments1% agarose2% agarose8Fragment Resolution

Konsentrasi gel (akan berpengaruh ke ukuran pori) akan mempengaruhi ukuran DNA yang di separasi9Purposes for Agarose Gel ElectrophoresisMenganalisis ukuran molekulMemisahkan suatu molekulMengkuantifikasi molekul10Procedure11Components of an Electrophoresis SystemPower supply and chamber, a source of power supplyBuffer, a fluid mixture of water and ions Agarose gel, a porous material that molecules migrates throughGel casting materials12Ions: atoms that have a positive or negative charge because they have lost or gained electrons.Electrophoresis: migration of ions at different speeds is a basic principal

BufferDyesPower Supply+-CathodeAnode13During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic. The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).

Casting trayGel combsPower supplyGel tankCoverElectrical leads Electrophoresis Equipments14Electrophoresis BufferTAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) pH bufferTris Acetic acid provide ions to support conductivity and maintain pHEDTA, prevent brake down of moleculesConcentration affects DNA migrationUse of water will produce no migratonHigh buffer conc. could melt the agarose gel15A buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or weak base and one of their salts. Because a change in pH can alter the charge on a particle, it is important to use a buffer solution when separating during electrophoresis.Gel Preparation Loading the gelRunning the gelOverview of Agarose Gel Electrophoresis16

Agarose is a linear polymer extracted from seaweed.Agarose is a linear polymer extracted from seaweed.Gel Preparation 17

AgaroseBuffer SolutionCombine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.18

Agarose is insoluble at room temperature (left).The agarose solution is boiled until clear (right).Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.Melting the Agarose19

Gel casting tray & combs20

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Pouring the gelAllow the agarose solution to cool slightly (~60C) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.22

When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the comb. 23

Place the gel in the electrophoresis chamber.24

Loading the GelCarefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.25

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Running the Gel27Migration of molecules in AgaroseRate of migration of a molecule is inversely proportional to the log of its molecular weight

Distance 1 / log-MW 28Agarose Gel Electrophoresis# To make DNA fragments visible after electrophoresis, the DNA must be stained

# The favoriteethidium bromide When bound to DNA it fluoresces under ultraviolet light Quite sensitive # Two big problems UV light can damage your eyes----and many students dont wear safety googles!!!!! Ethidium bromide is a mutagen!!!!!

29% AgaroseDNA fragment, kb

0.530-1

0.712-0.8

1.010-0.5

1.27-0.4

1.53-0.2