What Is The Function Of The Dna Size Standard In The Gel?

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

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

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Pei Yun Lee

¹Section of Molecular, Prison cell, and Developmental Biology, University of California Los Angeles

John Costumbrado

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

Chih-Yuan Hsu

ⁱDepartment of Molecular, Cell, and Developmental Biology, University of California Los Angeles

Yong Hoon Kim

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

Abstruse

Agarose gel electrophoresis is the nigh constructive way of separating Dna fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits^two. During gelation, agarose polymers associate non-covalently and course a network of bundles whose pore sizes determine a gel'southward 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 slope centrifugation, which only provided an approximation of size. To carve up DNA using agarose gel electrophoresis, the Dna is loaded into pre-bandage wells in the gel and a current applied. The phosphate courage of the Dna (and RNA) molecule is negatively charged, therefore when placed in an electric field, Dna fragments volition drift to the positively charged anode. Because Dna has a compatible mass/charge ratio, Dna molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves frontwards and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of Dna molecule; 2) agarose concentration; 3) DNA conformation^v; iv) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized nether uv lite after staining with an appropriate dye. By post-obit this protocol, students should be able to: one. Empathise the mechanism past which Deoxyribonucleic acid fragments are separated inside a gel matrix 2. Sympathize how conformation of the Dna molecule will decide its mobility through a gel matrix 3. Place an agarose solution of appropriate concentration for their needs 4. Set an agarose gel for electrophoresis of DNA samples 5. Fix the gel electrophoresis apparatus and power supply 6. Select an advisable voltage for the separation of Deoxyribonucleic acid fragments 7. Sympathize the mechanism by which ethidium bromide allows for the visualization of Dna bands viii. Make up one's mind the sizes of separated Dna fragments

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

Protocol

1. Preparation of the Gel

1. Counterbalance out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a due west/v per centum solution. The concentration of agarose in a gel volition depend on the sizes of the Dna fragments to be separated, with nearly gels ranging between 0.5%-two%. The book of the buffer should not exist greater than ane/iii of the capacity of the flask.

2. Add running buffer to the agarose-containing flask. Swirl to mix. The nigh common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).

3. Cook the agarose/buffer mixture. This is nearly normally done by heating in a microwave, but can likewise be done over a Bunsen flame. At 30 due south 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.v μg/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-xxx min, followed past destaining in running buffer for an equal length of time.

Note: EtBr is a suspected carcinogen and must be properly tending of per institution regulations. Gloves should ever be worn when handling gels containing EtBr. Culling dyes for the staining of Dna are available; still EtBr remains the most popular one due to its sensitivity and price.

1. Allow the agarose to absurd either on the benchtop or past incubation in a 65 °C h2o bath. Failure to do so will warp the gel tray.

2. Place the gel tray into the casting apparatus. Alternatively, ane may also tape the open edges of a gel tray to create a mold. Identify an appropriate comb into the gel mold to create the wells.

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

ii. Setting up of Gel Apparatus and Separation of DNA Fragments

1. Add loading dye to the DNA samples to be separated (Fig. 2). Gel loading dye is typically made at 6X concentration (0.25% bromphenol blueish, 0.25% xylene cyanol, thirty% glycerol). Loading dye helps to track how far your Dna sample has traveled, and likewise allows the sample to sink into the gel.

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

3. Add enough running buffer to cover the surface of the gel. It is of import to use the aforementioned running buffer as the one used to prepare the gel.

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

5. Remove the lid. Slowly and advisedly load the Dna sample(s) into the gel (Fig. iii). An appropriate Deoxyribonucleic acid size marker should always exist loaded along with experimental samples.

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

7. Plough on the ability. Run the gel until the dye has migrated to an appropriate distance.

3. Observing Separated Deoxyribonucleic acid fragments

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

2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on newspaper towels to absorb whatsoever actress running buffer.

3. Remove the gel from the gel tray and expose the gel to uv lite. This is most commonly done using a gel documentation system (Fig. 4). DNA bands should show up as orange fluorescent bands. Take a picture of the gel (Fig. 5).

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

4. Representative Results

Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After separation, the resulting DNA fragments are visible as conspicuously divers 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 1.5% agarose gel forth with a 2-log DNA ladder.

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

Figure ii. A student adding loading dye to her DNA samples.

Figure three. A educatee loading the Dna sample into a well in the gel.

Figure four. An case of a gel documentation system.

Figure v. An paradigm of a gel post electrophoresis. EtBr was added to the gel before electrophoresis to a concluding concentration of 0.5 μg/ml, followed by separation at 100 V for one hour. The gel was exposed to uv low-cal and the movie taken with a gel documentation system.

Give-and-take

Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose'south high gel forcefulness allows for the handling of low percent gels for the separation of large Dna fragments. Molecular sieving is determined by the size of pores generated by 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 effective at the separation of Dna fragments betwixt 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will demand to employ pulse field gel electrophoresis^{half dozen}, which involves the application of alternating current from 2 different directions. In this way larger sized Dna fragments are separated by the speed at which they reorient themselves with the changes in current direction. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Dissimilar agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemic reaction. These thinner gels are of college concentration, are run vertically and have better resolution. In mod Deoxyribonucleic acid sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application of high voltages, thereby enabling the separation of DNA fragments (and the determination of Dna sequence) quickly.

Agarose can be modified to create low melting agarose through hydroxyethylation. Low melting agarose is more often than not used when the isolation of separated Deoxyribonucleic acid fragments is desired. Hydroxyethylation reduces the packing density of the agarose bundles, finer reducing their pore size⁸. This means that a DNA fragment of the same size volition have longer to motion through a depression melting agarose gel equally opposed to a standard agarose gel. Because the bundles associate with 1 another through non-covalent interactions⁹, information technology is possible to re-cook an agarose gel afterward it has fix.

EtBr is the most common reagent used to stain DNA in agarose gels^ten. When exposed to uv lite, electrons in the effluvious ring of the ethidium molecule are activated, which leads to the release of energy (low-cal) equally the electrons return to ground country. EtBr works past intercalating itself in the DNA molecule in a concentration dependent way. This allows for an estimation of the amount of Deoxyribonucleic acid in any particular DNA band based on its intensity. Considering of its positive charge, the use of EtBr reduces the Deoxyribonucleic acid migration charge per unit by 15%. EtBr is a doubtable mutagen and carcinogen, therefore i must practise intendance when handling agarose gels containing it. In improver, EtBr is considered a hazardous waste matter and must exist tending of accordingly. Alternative stains for Dna in agarose gels include SYBR Gold, SYBR dark-green, Crystal Violet and Methyl Blueish. Of these, Methyl Blue and Crystal Violet practise not require exposure of the gel to uv calorie-free for visualization of Dna bands, thereby reducing the probability of mutation if recovery of the Deoxyribonucleic acid fragment from the gel is desired. However, 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 culling dyes either cannot be or exercise non work well when added directly to the gel, therefore the gel will have to exist post stained later on electrophoresis. Because of cost, ease of use, and sensitivity, EtBr all the same remains the dye of choice for many researchers. However, in certain situations, such as when hazardous waste disposal is hard or when young 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 density to the sample, allowing 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, allowing for the estimation of the altitude that DNA fragments have migrated.

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

In decision, since the adoption of agarose gels in the 1970s for the separation of Deoxyribonucleic acid, information technology has proven to be one of the virtually useful and versatile techniques in biological sciences research.

Disclosures

We have goose egg to disclose.

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What Is The Function Of The Dna Size Standard In The Gel?,

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

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