Agarose Gel Electrophoresis Aim: To perform agarose gel electrophoresis by loading the DNA samples. Introduction: DNA can be electrophoresed through gel prepared by melting and re-gelling agarose. Agarose gel electrophoresis is usefitl in identification, separation of DNA molecule between the size of 500 — 2500 bp and subsequent purification of DNA molecule of interest in a heterogeneous mixture. Inclusion of a series of size standards on a gel means that the size of a DNA fragment can be determined from its distance of migration. Resolution of DNA species on Agarose gels made it a widely used reliable method in gene manipulation experiments and is a rapid and relatively inexpensive method. If stained with ethidium bromide, florescent substances, 1 — 10 ng of DNA could be visualized using a light source, Principle: Agarose is a copolymer of D-galactose and 3, 6, anhydro L- galactose. It forms a gel by hydrogen bonding and pore size depends on the agarose concentration. The movement of DNA fragments within the gel matrix is influenced by agarose concentration. Low agarose concentration improves the resolution of large fragments, but reduces the resolution of smaller fragment and vice versa. The DNA molecule are separated by electrophoresis on the basis of their molecular size between 500 — 2500 bp small pieces of DNA migrate through the gel matrix faster than larger pieces under the influence of electric current, shape or the confirmation of the molecule and magnitude of net charges on the molecule or the applied current (DNA molecules have a net negative charge and migrate towards the anode). Agarose gel electrophoresis is conducted in a horizontal configuration; there is less distortion (collapse) during electrophoresis and the bands of DNA are less distorted. The easiest gel system to operate seems to be one that has the gel completely submerged by only one millimetre in the electrophoresis buffer. The ethidium bromide dye intercalated between the bases of DNA molecule and fluoresces bright orange when irradiated with UV light.Material required: Horizontal gel electrophoresis apparatus, Gel casting platform, gel combs (slot formers) , DC power supply unit, Microwave Oven, UV Tran illuminator, Micropipettes, Para film, Adhesive tapes, Eppendorf tubes, tips, gel scoop, Glass wares, Tris Borate - EDTA (TBE), Agarose, DNA molecular weight markers. Ethidium Bromide solution: issolve 0.2 gin 20 ml distilled water, mix well store at 4°C) Sample buffer / loading buffer 10X, 40% Sucrose, 0.25% Bromophenol blue(All w/v in 1 X TBE) Procedure: 1 gm of agarose was weighed and transferred to a conical flask. To the flask 100 ml of 1X TBE buffer was added. . The agarose was melted completely into a clear solution, using a microwave oven. The molten agarose was cooled to approximately 40°C and ethidium bromide was added to a final concentration of 4 11 of solution of ethidium bromide to 100 ml gel mixture and mixed well. . The molten agarose was poured in a pre-set template with well forming comb (in advance, an agarose gel template was cleaned with 70% ethanol and sealed its end with adhesive tapes). - Placed horizontally on a levelling table and an appropriate well-forming comb was placed. If the gel plate is not horizontal, wedge-shaped gel will form and DNA fragments will migrate peculiarly within it. The teeth of comb should not touch the bottom of the plate leaving at least 1 mm between comb and the gel plate while pouring. |. The electrophoresis chamber was filled with 0.5X TEB buffer until the gel is covered by a couple of millimetres, Gently lift the comb to avoid damage of the wells, the gel is now ready for loading. . To perform electrophoresis Sit] of gel loading dye was added to the sample and mix well by pipetting, 15 pl of the samples were loaded carefully with a pipette by slowly expelling thesolution into a well, with the pipette tip slightly below the top of the well. Do not hold the pipette too far in the well: the sample will sometimes come out the bottom. Try not to plug the pipette tip against the side of the well either: the sample will usually squirt out when there gets to be enongh pressure from the pipette. 7, The appropriate marker DNA flanking the sample lanes was loaded. 8. The lid on the electrophoresis kit was closed. The DNA was mun toward the red (+) terminal Electrophoresis the gel to voltage of 50 V or 100 V for a couple of hours. 9. Stop electrophoresis by tuming off the power supply, when the dye, Bromophenol blue has migrated a distance judged sufficient for separation of the DNA fragments. Visualize DNA band(s) on an UV Tran-illuminator. Observation: AGAROSE GEL ELECTROPHORESISNote: The above procedure is for making 100 ml of 1% agarose gel. Depending on the nature of DNA sample to be analyzed and the volume of the template, the experimenter should decide the percentage and the volume of the gel. For instance, plasmid DNA could be analyzed on 0.6 — 0.8 % gel. For analyzing chromosomal DNA, 0.4% gel is preferable. However, as the handling of 0.4% gel is difficult, chromosomal DNA can be analyzed on a higher percentage gel. For analyzing PCR products 1.5 — 2.0 % gel is required. Agarose % Range of separation | 15 200 - 3000 1.2 400 - 7000 1.0 500 — 10000 07 "800 — 12000 0.5 1000 — 30000