How Do You Know Which Lane Has the Largest Dna Fragment on the Gel

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

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Pei Yun Lee

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

John Costumbrado

^oneSection of Molecular, Jail cell, and Developmental Biological science, University of California Los Angeles

Chih-Yuan Hsu

^oneSection of Molecular, Cell, and Developmental Biology, University of California Los Angeles

Yong Hoon Kim

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

Abstract

Agarose gel electrophoresis is the most effective fashion of separating Dna fragments of varying sizes ranging from 100 bp to 25 kb^ane. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of Deoxyribonucleic acid. Prior to the adoption of agarose gels, Dna was primarily separated using sucrose density gradient centrifugation, which just provided an approximation of size. To separate Deoxyribonucleic acid using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a electric current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, Deoxyribonucleic acid fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size inside 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 move through an agarose gel is "biased reptation", whereby the leading edge moves forwards and pulls the rest of the molecule along^iv. The charge per unit of migration of a DNA molecule through a gel is determined by the post-obit: i) size of Deoxyribonucleic acid molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, five) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light afterward staining with an advisable dye. By post-obit this protocol, students should be able to: ane. Understand the mechanism by which Deoxyribonucleic acid fragments are separated within a gel matrix 2. Empathize how conformation of the Dna molecule volition determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of Dna samples 5. Ready up the gel electrophoresis apparatus and power supply 6. Select an advisable voltage for the separation of DNA fragments 7. Sympathize the mechanism by which ethidium bromide allows for the visualization of Deoxyribonucleic acid bands 8. Make up one's mind the sizes of separated DNA fragments

Keywords: Genetics, Result 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 w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the Dna fragments to exist separated, with almost gels ranging between 0.5%-2%. The volume of the buffer should non be greater than 1/iii of the capacity of the flask.

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

3. Melt the agarose/buffer mixture. This is most usually done past heating in a microwave, but can also exist washed over a Bunsen flame. At thirty s intervals, remove the flask and swirl the contents to mix well. Echo until the agarose has completely dissolved.

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

Note: EtBr is a suspected carcinogen and must exist properly disposed of per establishment regulations. Gloves should e'er be worn when treatment gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular 1 due to its sensitivity and toll.

1. Permit the agarose to absurd either on the benchtop or by incubation in a 65 °C h2o bath. Failure to do and then will warp the gel tray.

2. Identify the gel tray into the casting apparatus. Alternatively, ane may also tape the open up edges of a gel tray to create a mold. Identify an appropriate rummage 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 comb and place the gel in the gel box. Alternatively, the gel tin too be wrapped in plastic wrap and stored at 4 °C until utilise (Fig. 1).

2. Setting upward of Gel Apparatus and Separation of Dna Fragments

1. Add together loading dye to the Dna samples to be separated (Fig. 2). Gel loading dye is typically made at 6X concentration (0.25% bromphenol bluish, 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 ability supply to desired voltage (1-5V/cm betwixt electrodes).

3. Add plenty running buffer to cover the surface of the gel. It is important to apply the same 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 power supply are working.

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

6. Replace the hat to the gel box. The cathode (black leads) should be closer the wells than the anode (ruddy leads). Double check that the electrodes are plugged into the correct slots in the ability supply.

7. Turn on the ability. Run the gel until the dye has migrated to an appropriate altitude.

3. Observing Separated DNA fragments

1. When electrophoresis has completed, turn off the ability 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. Place the gel tray on paper towels to absorb any extra running buffer.

3. Remove the gel from the gel tray and betrayal the gel to uv low-cal. This is most commonly done using a gel documentation system (Fig. 4). Deoxyribonucleic acid bands should bear witness upward 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

Effigy 5 represents a typical result after 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 caste that allows for the useful decision 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 1. A solidified agarose gel later removal of the comb.

Effigy 2. A student calculation loading dye to her DNA samples.

Figure iii. A pupil loading the DNA sample into a well in the gel.

Figure four. An example of a gel documentation organization.

Effigy 5. An image of a gel post electrophoresis. EtBr was added to the gel before electrophoresis to a final concentration of 0.v μg/ml, followed by separation at 100 V for ane hour. The gel was exposed to uv light and the picture taken with a gel documentation system.

Discussion

Agarose gel electrophoresis has proven to exist an efficient and effective style of separating nucleic acids. Agarose's loftier gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined past 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 constructive at the separation of Dna fragments between 100 bp and 25 kb. To split up Dna fragments larger than 25 kb, 1 volition need to use pulse field gel electrophoresis^half-dozen, which involves the application of alternating electric current from two different directions. In this way larger sized Deoxyribonucleic acid 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 finer separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a gratuitous radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and have ameliorate resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The utilize of capillary tubes allows for the application of loftier voltages, thereby enabling the separation of DNA fragments (and the determination of DNA sequence) rapidly.

Agarose tin can exist modified to create depression 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 size^eight. This means that a Deoxyribonucleic acid fragment of the same size volition take longer to move through a low melting agarose gel as opposed to a standard agarose gel. Because the bundles associate with one some other through non-covalent interactions^ix, 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 energy (calorie-free) as the electrons return to basis state. EtBr works by intercalating itself in the Deoxyribonucleic acid molecule in a concentration dependent style. This allows for an interpretation of the amount of DNA in any particular DNA band based on its intensity. Because of its positive accuse, the use of EtBr reduces the DNA migration charge per unit by fifteen%. EtBr is a suspect mutagen and carcinogen, therefore one must practise care when handling agarose gels containing it. In addition, EtBr is considered a hazardous waste and must be disposed of accordingly. Alternative stains for Dna in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue. Of these, Methyl Blue and Crystal Violet do not 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. Nonetheless, their sensitivities are lower than that of EtBr. SYBR gilt and SYBR green are both highly sensitive, uv dependent dyes with lower toxicity than EtBr, only they are considerably more expensive. Moreover, all of the alternative dyes either cannot be or do not work well when added straight to the gel, therefore the gel volition take to exist post stained later electrophoresis. Considering of cost, ease of utilise, and sensitivity, EtBr still remains the dye of selection for many researchers. Even so, in certain situations, such as when chancy waste material disposal is difficult 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, assuasive information technology to sink into the gel. Second, the dyes provide color and simplify the loading process. Finally, the dyes movement at standard rates through the gel, assuasive for the estimation of the distance that DNA fragments have migrated.

The exact sizes of separated DNA fragments can be adamant past plotting the log of the molecular weight for the different bands of a Dna standard confronting the distance traveled by each band. The DNA standard contains a mixture of Deoxyribonucleic acid fragments of pre-determined sizes that can exist compared against the unknown Dna samples. It is important to annotation 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 by a linear Deoxyribonucleic acid fragment of the same size, with the open circular course 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 enquiry.

Disclosures

Nosotros have nothing to disclose.

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

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