What Technique Separates Dna Fragments By Size

• Periodical List
• J Vis Exp
• (62); 2012
• PMC4846332

J Vis Exp. 2012; (62): 3923.

Agarose Gel Electrophoresis for the Separation of Deoxyribonucleic acid Fragments

Pei Yun Lee

¹Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles

John Costumbrado

¹Section of Molecular, Jail cell, and Developmental Biological science, Academy of California Los Angeles

Chih-Yuan Hsu

¹Department of Molecular, Cell, and Developmental Biology, Academy of California Los Angeles

Yong Hoon Kim

^aneDepartment of Molecular, Jail cell, and Developmental Biology, University of California Los Angeles

Abstract

Agarose gel electrophoresis is the most constructive manner of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb^one. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (50- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and course a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate Deoxyribonucleic acid using agarose gel electrophoresis, the Dna is loaded into pre-bandage wells in the gel and a electric current practical. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, Deoxyribonucleic acid fragments will drift to the positively charged anode. Considering DNA has a uniform mass/accuse ratio, Deoxyribonucleic acid molecules are separated by size within an agarose gel in a blueprint such that the distance traveled is inversely proportional to the log of its molecular weightⁱⁱⁱ. The leading model for Dna movement through an agarose gel is "biased reptation", whereby the leading border moves forward and pulls the residue of the molecule along^four. The rate of migration of a DNA molecule through a gel is determined by the post-obit: one) size of DNA molecule; 2) agarose concentration; 3) Deoxyribonucleic acid conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) blazon of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can exist visualized under uv light subsequently staining with an appropriate dye. By following this protocol, students should exist able to: 1. Sympathize the machinery by which DNA fragments are separated within a gel matrix ii. Understand how conformation of the DNA molecule will make up one's mind its mobility through a gel matrix iii. Identify an agarose solution of appropriate concentration for their needs four. Prepare an agarose gel for electrophoresis of DNA samples five. Set the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of Dna bands viii. Determine the sizes of separated Deoxyribonucleic acid fragments

Keywords: Genetics, Issue 62, Gel electrophoresis, agarose, Deoxyribonucleic acid separation, ethidium bromide

Protocol

1. Grooming of the Gel

1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/five percentage solution. The concentration of agarose in a gel will depend on the sizes of the Dna fragments to exist separated, with nearly gels ranging between 0.5%-ii%. The volume of the buffer should not exist greater than one/3 of the capacity of the flask.

2. Add running buffer to the agarose-containing flask. Swirl to mix. The near mutual gel running buffers are TAE (xl mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, i mM EDTA).

3. Melt the agarose/buffer mixture. This is most usually done by heating in a microwave, merely can likewise exist washed over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.

4. Add together ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may also be stained afterwards electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-thirty min, followed by destaining in running buffer for an equal length of time.

Notation: EtBr is a suspected carcinogen and must be properly disposed of per establishment regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; nonetheless EtBr remains the most popular 1 due to its sensitivity and cost.

1. Allow the agarose to cool either on the benchtop or past incubation in a 65 °C water bath. Failure to do so will warp the gel tray.

2. Place the gel tray into the casting apparatus. Alternatively, ane may likewise record the open edges of a gel tray to create a mold. Place an advisable comb into the gel mold to create the wells.

3. Cascade the molten agarose into the gel mold. Allow the agarose to ready at room temperature. Remove the rummage and place the gel in the gel box. Alternatively, the gel tin can also be wrapped in plastic wrap and stored at 4 °C until utilise (Fig. one).

two. Setting upwards of Gel Apparatus and Separation of Deoxyribonucleic acid Fragments

1. Add loading dye to the Deoxyribonucleic acid samples to exist separated (Fig. ii). Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.

2. Program the power supply to desired voltage (1-5V/cm between electrodes).

3. Add plenty running buffer to cover the surface of the gel. It is important to use the aforementioned running buffer as the ane used to prepare the gel.

4. Attach the leads of the gel box to the power supply. Plow on the power supply and verify that both gel box and ability supply are working.

5. Remove the lid. Slowly and carefully load the DNA sample(s) into the gel (Fig. 3). An appropriate Dna size mark should always be loaded forth with experimental samples.

6. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the anode (blood-red leads). Double check that the electrodes are plugged into the correct slots in the power supply.

7. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

3. Observing Separated Dna fragments

1. When electrophoresis has completed, turn off the power supply and remove the lid of the gel box.

2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Identify the gel tray on paper towels to absorb any actress running buffer.

3. Remove the gel from the gel tray and expose the gel to uv calorie-free. This is virtually normally done using a gel documentation system (Fig. 4). DNA bands should evidence up equally orangish fluorescent bands. Take a motion-picture show of the gel (Fig. 5).

4. Properly dispose of the gel and running buffer per institution regulations.

4. Representative Results

Effigy 5 represents a typical result subsequently agarose gel electrophoresis of PCR products. After separation, the resulting DNA fragments are visible as clearly defined bands. The Dna standard or ladder should be separated to a degree that allows for the useful determination of the sizes of sample bands. In the example shown, Dna fragments of 765 bp, 880 bp and 1022 bp are separated on a 1.five% agarose gel along with a 2-log DNA ladder.

Figure 1. A solidified agarose gel after removal of the rummage.

Effigy two. A student adding loading dye to her DNA samples.

Figure 3. A student loading the Dna sample into a well in the gel.

Effigy 4. An example of a gel documentation system.

Effigy 5. An image of a gel post electrophoresis. EtBr was added to the gel earlier electrophoresis to a final concentration of 0.5 μg/ml, followed by separation at 100 V for 1 hr. The gel was exposed to uv lite and the picture taken with a gel documentation system.

Discussion

Agarose gel electrophoresis has proven to be an efficient and constructive way of separating nucleic acids. Agarose'southward high gel forcefulness allows for the handling of low percentage gels for the separation of big DNA fragments. Molecular sieving is determined by the size of pores generated past the bundles of agarose⁷ in the gel matrix. In general, the higher the concentration of agarose, the smaller the pore size. Traditional agarose gels are most constructive at the separation of DNA fragments between 100 bp and 25 kb. To split Deoxyribonucleic acid fragments larger than 25 kb, one volition need to use pulse field gel electrophoresis^six, which involves the application of alternating current from two different directions. In this style larger sized Dna fragments are separated by the speed at which they reorient themselves with the changes in current management. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and take amend resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The employ of capillary tubes allows for the application of high voltages, thereby enabling the separation of Dna fragments (and the determination of Deoxyribonucleic acid sequence) quickly.

Agarose tin be modified to create low melting agarose through hydroxyethylation. Depression melting agarose is generally used when the isolation of separated DNA fragments is desired. Hydroxyethylation reduces the packing density of the agarose bundles, effectively reducing their pore size⁸. This means that a DNA fragment of the same size will have longer to move through a low melting agarose gel every bit opposed to a standard agarose gel. Considering the bundles acquaintance with one another through non-covalent interactions⁹, it is possible to re-melt an agarose gel after it has set.

EtBr is the well-nigh common reagent used to stain Dna in agarose gels¹⁰. When exposed to uv lite, electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of free energy (light) as the electrons return to ground state. EtBr works by intercalating itself in the Dna molecule in a concentration dependent mode. This allows for an interpretation of the amount of Deoxyribonucleic acid in any detail Deoxyribonucleic acid band based on its intensity. Because of its positive accuse, the use of EtBr reduces the Deoxyribonucleic acid migration rate past 15%. EtBr is a suspect mutagen and carcinogen, therefore one must practise intendance when handling agarose gels containing it. In improver, EtBr is considered a hazardous waste and must be disposed of appropriately. Alternative stains for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blueish. Of these, Methyl Blue and Crystal Violet do non crave exposure of the gel to uv light for visualization of DNA bands, thereby reducing the probability of mutation if recovery of the DNA fragment from the gel is desired. Even so, their sensitivities are lower than that of EtBr. SYBR gold and SYBR green are both highly sensitive, uv dependent dyes with lower toxicity than EtBr, but they are considerably more expensive. Moreover, all of the alternative dyes either cannot be or practise not work well when added directly to the gel, therefore the gel will have to be postal service stained afterwards electrophoresis. Because of cost, ease of use, and sensitivity, EtBr even so remains the dye of selection for many researchers. Still, in certain situations, such every bit when hazardous waste product disposal is difficult or when immature students are performing an experiment, a less toxic dye may be preferred.

Loading dyes used in gel electrophoresis serve three major purposes. First they add together density to the sample, assuasive it to sink into the gel. Second, the dyes provide color and simplify the loading process. Finally, the dyes move at standard rates through the gel, assuasive for the estimation of the altitude that Deoxyribonucleic acid fragments have migrated.

The exact sizes of separated Dna fragments tin can be determined by plotting the log of the molecular weight for the different bands of a Deoxyribonucleic acid standard against the altitude traveled by each band. The DNA standard contains a mixture of Dna fragments of pre-determined sizes that can exist compared against the unknown Deoxyribonucleic acid samples. It is important to notation that different forms of Dna move through the gel at unlike rates. Supercoiled plasmid DNA, because of its compact conformation, moves through the gel fastest, followed by a linear Deoxyribonucleic acid fragment of the same size, with the open round form traveling the slowest.

In conclusion, since the adoption of agarose gels in the 1970s for the separation of DNA, it has proven to exist ane of the most useful and versatile techniques in biological sciences research.

Disclosures

We have nix to disembalm.

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What Technique Separates Dna Fragments By Size,

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/

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