agarose gel experiment

Agarose Gel Electrophoresis: Principle, Procedure, Results

Agarose gel electrophoresis is one of the most common electrophoresis techniques which is relatively simple and straightforward to perform but possesses great resolving power. The agarose gel consists of microscopic pores that act as a molecular sieve that separates molecules based on the charge, size, and shape.

Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes, and it is a convenient analytical method for separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis whereas pulse-field gel electrophoresis is used to separate DNA fragments larger than 25 kb.  Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins.

Table of Contents

The separation medium is a gel made from agarose. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties. In general, the higher the concentration of agarose, the smaller the pore size.

To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current is applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size.

Factors affecting the migration of DNA

Agarose concentration:.

The mobility of DNA molecules is inversely proportional to gel concentration. Higher percentage gels are sturdier and easier to handle but the mobility of molecules and staining will take longer because of the tighter matrix of the gel. The most common agarose gel concentration for separating dyes or DNA fragments is 0.8% . However, some experiments require agarose gels with a higher percentage, such as 1% or 1.5%.

Size of DNA molecule

The sieving properties of the agarose gel influence the rate at which a molecule migrates. The separation occurs because smaller molecules pass through the pores of the gel more easily than larger ones. If the size of the two fragments is similar or identical, they will migrate together in the gel.

DNA conformation

Different forms of DNA move through the gel at different rates; DNA molecules having a more compact shape (e.g. plasmid DNA ) moves faster through the gel compared with linear DNA fragments of the same size. The migration rate of linear fragments of DNA is inversely proportional to log 10 of their size in base pairs. This means that the smaller the linear fragment, the faster it migrates through the gel.

Applied voltage

Mobility of DNA molecules is also affected by the applied voltage. Within a range, the higher the applied voltage, the faster the sample migration.

Preparation of Agarose gel matrix

The centerpiece of agarose gel electrophoresis is the horizontal gel electrophoresis apparatus. The gel is made by dissolving agarose powder in a boiling buffer solution.

The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The solution is then cooled to approximately 55°C and poured into a casting tray which serves as a mold. A well-former template (often called a comb) is placed across the end of the casting tray to form wells when the gel solution solidifies.

After the gel solidifies, the gel is submerged in a buffer-filled electrophoresis chamber which contains a positive electrode (anode) at one end, and a negative electrode (cathode) at the other.  The volume of the buffer should not be greater than 1/3 of the electrophoresis chamber. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).

Sample preparation and loading

Samples are prepared for electrophoresis by mixing them with loading dyes. Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dyes used in gel electrophoresis serve three major purposes:

• add density to the sample, allowing it to sink into the gel.
• provide color and simplify the loading process.
• the dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated.

These samples are delivered to the sample wells with a clean micropipette (variable automatic micropipette is the preferred one).

Ethidium bromide can be added to the gel during this step or alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.

Applying electric current and separating biomolecules

A direct current (D.C.) power source is connected to the electrophoresis apparatus and an electrical current is applied.  Charged molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). The buffer serves as a conductor of electricity and controls the pH, which is important to the charge and stability of biological molecules. Since DNA has a strong negative charge at neutral pH, it migrates through the gel towards the positive electrode during electrophoresis.

The bluish-purple dye allows for visual tracking of sample migration during electrophoresis. The gel is run until the dye has migrated to an appropriate distance.

Visualization

The agarose gel will have to be post-stained after electrophoresis. The most commonly used stain for visualizing DNA is ethidium bromide (EtBr)*. Alternative stains for DNA in agarose gels include SYBR Gold, SYBR green, crystal violet, and methyl blue. The sensitivities of methylene blue and crystal violet are low compared with ethidium bromide. SYBR gold and SYBR green are highly sensitive but more expensive than EtBr.

EtBr works by intercalating itself in the DNA molecule in a concentration-dependent manner. When exposed to a short wave ultraviolet light source (transilluminator), electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to the ground state. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity.

*Ethidium bromide is a suspect mutagen and carcinogen so must be handled cautiously. It is a hazardous waste so must be disposed of according to strict local and/or state guidelines. Stains containing methylene blue are considered safer than ethidium bromide, but should still be handled and disposed with care.  

The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard (DNA ladder) against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples.

DNA concentrations can be estimated by:

A. Taking absorbance at 260 nm. At 260 nm, an absorbance (A) of 1 unit corresponds to a concentration of:

• 50 μg/ml for dsDNA
• 40 μg/ml for RNA
• 33 μg/ml for ssDNA
• 20-30 µg/ml for oligonucleotides

Although this method is quick and nondestructive and gives information about the purity of the sample (e.g., presence of protein or organic contaminants), reliable estimates are obtained only with concentrations of at least 1 μg/ml. Additionally, this method cannot distinguish between DNA and RNA.

B. Intensity of Ethidium Bromide Fluorescence:

The amount of DNA in a sample can be estimated from the intensity of ethidium bromide fluorescence (fluorescence emitted by ethidium bromide is proportional to the amount of DNA). The DNA quantity in an “unknown” solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA of similar size. This method is useful if a DNA sample is contaminated with other compounds that absorb in the UV range or is too dilute to measure at 260 nm.

References and further reading

• Agarose Gel Electrophoresis for the Separation of DNA Fragments. Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim J Vis Exp. 2012; (62): 3923.
• Principles and Practice of Agarose Gel Electrophoresis: EDVOTEK
• Agarose gel electrophoresis: Rice University
• Agarose gel electrophoresis of DNA – Principle, Protocol and Uses: Laboratoryinfo.com

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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Agarose Gel Electrophoresis for the Separation of DNA Fragments

Pei yun lee, john costumbrado, chih-yuan hsu, yong hoon kim.

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Correspondence to: Pei Yun Lee at [email protected]

Collection date 2012.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb 1 . Agarose is isolated from the seaweed genera Gelidium and Gracilaria , and consists of repeated agarobiose (L- and D-galactose) subunits 2 . 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 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 DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform 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 weight 3 . The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along 4 . 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 5 ; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will 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. Set up 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 8. Determine the sizes of separated DNA fragments  

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

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1. Preparation of the Gel

Weigh 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 be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.

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

Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done 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.

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 15-30 min, followed by destaining in running buffer for an equal length of time.

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

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

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

Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be wrapped in plastic wrap and stored at 4 °C until use ( Fig. 1 ).

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

Add loading dye to the DNA samples to be separated ( Fig. 2 ). 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.

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

Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel.

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.

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

Replace 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 correct slots in the power supply.

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

3. Observing Separated DNA fragments

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

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.

Remove the gel from the gel tray and expose the gel to uv light. 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 ).

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 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 1.5% agarose gel along with a 2-log DNA ladder.

Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose's high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of agarose 7 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 between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis 6 , which involves the application of alternating current from two 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. 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 have better resolution. In modern DNA 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 generally used when the isolation of separated DNA fragments is desired. Hydroxyethylation reduces the packing density of the agarose bundles, effectively reducing their pore size 8 . This means that a DNA fragment of the same size will take longer to move through a low melting agarose gel as opposed to a standard agarose gel. Because the bundles associate with one another through non-covalent interactions 9 , it is possible to re-melt an agarose gel after it has set.

EtBr is the most common reagent used to stain DNA in agarose gels 10 . When exposed to uv light, electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to ground state. EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity. Because of its positive charge, the use of EtBr reduces the DNA migration rate by 15%. EtBr is a suspect mutagen and carcinogen, therefore one must exercise care when handling agarose gels containing it. In addition, 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 Blue. Of these, Methyl Blue and Crystal Violet do not require 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. 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 alternative dyes either cannot be or do not work well when added directly to the gel, therefore the gel will have to be post stained after electrophoresis. Because of cost, ease of use, and sensitivity, EtBr still remains the dye of choice for many researchers. However, in certain situations, such as when hazardous waste 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, 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 distance that DNA fragments have migrated.

The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can 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 by a linear DNA fragment of the same size, with the open circular form traveling the slowest.

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

Disclosures

We have nothing to disclose.

• Sambrook J, Russell DW. Molecular Cloning. 3rd 2001.
• Kirkpatrick FH. Overview of agarose gel properties. Electrophoresis of large DNA molecules: theory and applications. 1991. pp. 9–22.
• Helling RB, Goodman HM, Boyer HW. Analysis of endonuclease R•EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 1974;14:1235–1244. doi: 10.1128/jvi.14.5.1235-1244.1974. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Smith SB, Aldridge PK, Callis JB. Observation of individual DNA molecules undergoing gel electrophoresis. Science. 1989;243:203–206. doi: 10.1126/science.2911733. [ DOI ] [ PubMed ] [ Google Scholar ]
• Aaji C, Borst P. The gel electrophoresis of DNA. Biochim. Biophys. Acta. 1972;269:192–200. doi: 10.1016/0005-2787(72)90426-1. [ DOI ] [ PubMed ] [ Google Scholar ]
• Lai E, Birren BW, Clark SM, Simon MI, Hood L. Pulsed field gel electrophoresis. Biotechniques. 1989;7:34–42. [ PubMed ] [ Google Scholar ]
• Devor EJ. IDT tutorial: gel electrophoresis. 2010. Available from: http://cdn.idtdna.com/Support/Technical/TechnicalBulletinPDF/Gel_Electrophoresis.pdf .
• Serwer P. Agarose gels: properties and use for electrophoresis. Electrophoresis. 1983;4:375–382. [ Google Scholar ]
• Dea ICM, McKinnon AA, Rees DA. Tertiary and quaternary structure and aqueous polysaccharide systems which model cell wall adhesion: reversible changes in conformation and association if agarose, carrageenan and galactomannans. J. Mol. Biol. 1972;68:153–172. doi: 10.1016/0022-2836(72)90270-7. [ DOI ] [ PubMed ] [ Google Scholar ]
• Sharp PA, Sugden B, Sambrook J. Detection of two restriction endonuclease activities in H. parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry. 1973;12:3055–3063. doi: 10.1021/bi00740a018. [ DOI ] [ PubMed ] [ Google Scholar ]
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Microbe Notes

Agarose Gel Electrophoresis: Principle, Parts, Steps, Uses

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA , RNA or proteins in a matrix of agarose.

• Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool.
• They are the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation.

Table of Contents

Interesting Science Videos

Principle of Agarose Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size in a solid support medium such as an agarose gel. Sample (DNA) are pipetted into the sample wells, followed by the application of an electric current which causes the negatively-charged DNA to migrate (electrophorese) towards the anodal, positive (+ve) end. The rate of migration is proportional to size: smaller fragments move more quickly and wind up at the bottom of the gel.

DNA is visualized by including in the gel an intercalating dye, ethidium bromide. DNA fragments take up the dye as they migrate through the gel. Illumination with ultraviolet light causes the intercalated dye to fluoresce.

The larger fragments fluoresce more intensely. Although each of the fragments of a single class of molecule is present in equimolar proportions, the smaller fragments include less mass of DNA, take up less dye, and therefore fluoresce less intensely. A “ladder” set of DNA fragments of known size can be run simultaneously and used to estimate the sizes of the other unknown fragments.

Requirements/ Instrumentation of Agarose Gel Electrophoresis

The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:

• An electrophoresis chamber and power supply
• Gel casting trays , which are available in a variety of sizes and composed of UVtransparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
• Sample combs , around which molten medium is poured to form sample wells in the gel.
• Electrophoresis buffer , usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
• Loading buffer , which contains something dense (e.g. glycerol) to allow the sample to “fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
• Staining : DNA molecules are easily visualized under an ultraviolet lamp when electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively, nucleic acids can be stained after electrophoretic separation by soaking the gel in a solution of ethidium bromide. When intercalated into doublestranded DNA, fluorescence of this molecule increases greatly. It is also possible to detect DNA with the extrinsic fluor 1-anilino 8-naphthalene sulphonate.
• Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in gels.

Steps Involved in Agarose Gel Electrophoresis

• To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, and heated in a microwave oven to melt it.

The concentration of Agarose Gel

• The percentage of agarose used depends on the size of fragments to be resolved.
• The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.2% to 3%.
• The lower the concentration of agarose, the faster the DNA fragments migrate.
• In general, if the aim is to separate large DNA fragments, a low concentration of agarose should be used, and if the aim is to separate small DNA fragments, a high concentration of agarose is recommended.
• Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to facilitate visualization of DNA after electrophoresis.
• After cooling the solution to about 60oC, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature.
• After the gel has solidified, the comb is removed, taking care not to rip the bottom of the wells.
• The gel, still in plastic tray, is inserted horizontally into the electrophoresis chamber and is covered with buffer.
• Samples containing DNA mixed with loading buffer are then pipetted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied.
• The current flow can be confirmed by observing bubbles coming off the electrodes.
• DNA will migrate towards the positive electrode, which is usually colored red, in view of its negative charge.
• The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes like bromophenol blue and xylene cyanol dyes.

Agarose Gel Electrophoresis Video Animation

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA.

• Estimation of the size of DNA molecules
• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
• Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis.
• The agarose gel electrophoresis is widely employed to estimate the size of DNA fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned DNA.
• Agarose gel electrophoresis is commonly used to resolve circular DNA with different supercoiling topology, and to resolve fragments that differ due to DNA synthesis.
• In addition to providing an excellent medium for fragment size analyses, agarose gels allow purification of DNA fragments. Since purification of DNA fragments size separated in an agarose gel is necessary for a number molecular techniques such as cloning, it is vital to be able to purify fragments of interest from the gel.

Advantages of Agarose Gel Electrophoresis

• For most applications, only a single-component agarose is needed and no polymerization catalysts are required. Therefore, agarose gels are simple and rapid to prepare.
• The gel is easily poured, does not denature the samples.
• The samples can also be recovered.

Disadvantages of Agarose Gel Electrophoresis

• Gels can melt during electrophoresis.
• The buffer can become exhausted.
• Different forms of genetic material may run in unpredictable forms.
• https://pdfs.semanticscholar.org/36cf/d4ada922c44d233b6ebfa2af2c956c92e4ec.pdf
• https://www.mun.ca/biology/scarr/Gel_Electrophoresis.html
• https://www.wou.edu/las/physci/ch462/Gel%20Electrophoresis.pdf
• https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
• https://msu.edu/course/css/451/Lecture/PT-electrophoresis%20(2009).pdf
• http://library.umac.mo/ebooks/b28050459.pdf

About Author

Sagar Aryal

8 thoughts on “Agarose Gel Electrophoresis: Principle, Parts, Steps, Uses”

understood most of it, althought the concept is little difficult but i saw video and read the full notes many times, it helped….. ThANK YOU

Satisfactory… Thanks uh a lot ????

Very informative and easy to understand.

Informative notes in easy language ????

Thank You for the Notes.. It’s very informative notes which have coverd allmost all the important things of AGE.

But I have a small doubt regarding the lines in “Principle Part”

Since DNA is negatively charged (anion) and it moves to Anode (+ve charged) end, not the Cathodal end, you mentioned in the paragraph of “Principle of Agarose Gel Electrophoresis”.

Please correct me if I am wrong.

Hi Mahesh, Thanks for the correction. I have updated the post with an anodal, positive (+ve) end. Best, Sagar

very informative notes and accurate easy to understand..keep doing like this..

This helped out in more clarification! Just had a lecture on electrophoresis today,with this I comprehend more on it. Thank you for this!

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Laboratory Notes

Protocol: Preparation of Agarose Gel for DNA Analysis

Agarose is insoluble in aqueous electrophoresis buffers at room temperature. However, when the suspension of agarose in an aqueous buffer (e.g., TAE or TBE) is heated to boiling, agarose particles melt and form uniform clear viscous solution. When this solution is allowed to cool down, it forms a translucent gel that has sieving properties and it allows separation of large macromolecules such as DNA, RNA and large proteins. 

When you plan to prepare an agarose gel you should keep the following things in your mind

Size-range of DNA fragments to be analyzed : This will help you to determine the agarose percentage in the gel and type of electrophoresis buffer. 

High percentage agarose gel for separation of short DNA fragments and low percentage for separation of large DNA fragments.

TBE buffer is a better choice for separation of short DNA fragments whereas TAE is for the separation of large DNA fragments.

Number of samples to be analyzed: This will help you to determine the number of wells in the agarose gel and its width. 

Sample volume: The volume of wells should be sufficient enough to accommodate required volume of each sample. Well volume can be adjusted by choosing a comb with appropriate teeth size (width and length) and gel thickness. Gel thickness can be controlled by the volume of melton agarose.

Visualization procedure: You can add DNA staining dye in the gel while preparing it or you can stain the gel after electrophoresis.

Purpose of gel: Analytical or preparative. If the purpose is to elute the DNA from the gel, TAE buffer is a better choice than the TBE buffer.

• Applications of agarose gels  
• Agarose Gel  

REQUIREMENTS

Reagents and solutions ◊ Agarose ◊ Ethidium bromide solution (10 mg/ml in water) or any other suitable DNA staining dye ◊ Electrophoresis buffer (TAE or TBE Buffer) ◊ Deionized/distilled water Equipment and disposables ◊ Gel casting tray and combs ◊ Micropipettes and tips ◊ Gloves ◊ Measuring cylinder ◊ Microwave/Hot plate

Note: 1. Prepare the casting tray by sealing both open ends of the tray with tape (video). Pour water in the tray to check whether the tray is sealed properly and is not leaky. 2. Casting tray and electrophoresis appratus can be purchased from several suppliers. Now many suppliers offer casting trays that do not need to be sealed with tape and have a special set-up to accommodate molten agarose without leakage. You must follow the procedure described in the manual.

Preparation a 0.8 % agarose gel (gel size – 10 cm x 12 cm x 0.4 cm) in TAE buffer or TBE buffer

Notes 1. Depending on the size of DNA fragments to be resolved, one can choose the concentration (0.5 – 2%) of the agarose gel. Here we have taken an example to clearly describe the preparation process. 2. Use a low percentage of agarose gel to resolve high molecular weight DNA and high percentage to resolve low molecular weight DNA. 3. Use 0.5X TBE buffer or 1X TAE buffer for running and preparation of agarose gel. Always use the same running buffer in which the agarose gel is prepared.

Step 1: Weigh out 0.4 g agarose in a conical flask/bottle. Add 50 ml of 1X TAE buffer. Suspend the agarose by swirling the flask. Wait for 1 – 2 min to allow hydration of agarose particles.

Notes 1. To make 0.8 % agarose gel of size 10 cm (width) x 12 cm (length) x 0.4 cm (thickness), 50 ml solution is required. 2. The volume of the flask/bottle should be 3 – 4 times the volume of the agarose solution being prepared.

Tip The total gel volume varies depending on the size of the casting tray. Use the following formula to calculate the volume of agarose solution: Total volume of agarose solution = width of casting tray x length of casting tray x thickness of gel.

Precaution: Remember to add buffer not water. If you prepare agarose gel in water, DNA will not move in the gel when you start electrophoresis.

Step 2: Weigh the flask/bottle.

Note: Sometimes, there is a significant loss of water during the melting process, which depends on the melting procedure. You can calculate the loss of water by weighing the flask/bottle just before and after the melting process. Loss of water is significantly high when you prepare agarose suspension in a conical flask/beaker (no lid) and melt it in a microwave. Step 3: Melt the agarose in a microwave or hot plate until the solution becomes clear. While heating, swirl the flask occasionally.

Tip Heat the solution for several short intervals instead of boiling continuously. Continuous boiling can cause the solution to boil out of the flask.

Precaution Make sure that the melted agarose solution appears clear and transparent, devoid of any suspended particles of agarose. Melt it more if there are some suspended particles. Step 4: Weigh the flask/bottle again and make up the loss by adding deionized/distilled water (do not add buffer). Step 5: Cool the solution until the temperature reaches 55 – 60°C.

Tips 1. Swirl the flask occasionally to cool the solution evenly. 2. You can store the solution in a 60°C water bath to prevent overcooling of agarose. This can take 15 – 20 min. A casting tray can be prepared during this time. Step 6 (optional): Add Ethidium bromide to agarose solution Add 2.5 μl ethidium bromide in the solution. Mix by gentle swirling. Avoid air bubble formation.

Precautions 1. Ethidium bromide is carcinogenic. Use appropriate safety measures (wear latex gloves and lab coat) to avoid any harm. 2. Do not add Ethidium bromide when the solution is very hot. Step 7: Set the comb and pour the molten agarose solution into the casting tray ♦ Pour the molten agarose solution into the casting tray. ♦ Insert the comb at appropriate place.

Tips 1. One can place the comb before pouring the agarose solution into the casting tray. 2. Remove air bubbles with the help of pipette tip.

Precaution While inserting the comb, take care that the teeth of the comb should not touch the bottom of the casting tray. If it touches, the well will be like a hole and samples will leak out from the well. Step 8: Wait until agarose is solidified completely. Solidified agarose gel will appear milky white. Agarose gel is ready for use.

Note Agarose gel can be stored for a few days. To store the agarose gel, we recommend not to remove comb and tape. Dip the agarose gel in the TBE buffer so that it contains moisture. Seal the gel in a plastic wrap and store it in the cold room (4°C). Before starting electrophoresis, let it come to room temperature.

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Course info, instructors.

• Prof. Drew Endy
• Dr. Natalie Kuldell
• Dr. Neal Lerner
• Dr. Agi Stachowiak
• Prof. Angela M. Belcher
• Dr. Atissa Banuazizi

Departments

• Biological Engineering

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• Systems Engineering

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Laboratory fundamentals in biological engineering, module 1.2: agarose gel electrophoresis.

Modules: 1.1 | 1.2 | 1.3 | 1.4 | 1.5 | 1.6 | 1.7

Introduction

Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and retarded; the smaller molecules wind through the matrix more easily and travel further from the well. Molecules of the same size and charge migrate the same distance from the well and collect into a band.

DNA and RNA are negatively charged molecules due to their phosphate backbone, and they naturally travel toward the positive charge at the far end of the gel. They are typically examined with agarose gels. Proteins are composed of amino acids that can be positively, negatively or uncharged. To give proteins a uniformly negative charge, they are coated with a detergent, SDS, prior to running them on a gel. Protein samples are also boiled to remove any secondary structure that might make two molecules of the same size migrate differently. Polyacrylamide is the matrix commonly used to separate proteins. These gels are typically run vertically while agarose gels are run horizontally but gravity has nothing to do with the separation.

Diagram of agarose gel setup, for agarose gel electrophoresis. (Figure by MIT OpenCourseWare.)

Today you will separate DNA fragments using an agarose matrix. Agarose is a polymer that comes from seaweed and if you’ve ever made Jell-O™, then you already have all the skills for pouring an agarose gel. To prepare these gels, agarose and buffer are microwaved until the agarose is melted. The molten agar is then poured into a horizontal casting tray, and a comb is added. Once the agar has solidified, the comb is removed, leaving wells into which the DNA sample can be loaded.

The distance a DNA fragment travels is inversely proportional to its length. Over time fragments of similar length accumulate into “bands” in the gel. Higher concentrations of agarose can be used to resolve smaller DNA fragments. This figure shows the same DNA fragments resolved with three agarose concentrations. The 1000 base pair fragment is indicated in each.

DNA fragments resolved with three agarose concentrations. The line indicates the 1000 base pair fragment. (Figure by MIT OpenCourseWare.)

Size vs. migration distance.

Ethidium Bromide is a fluorescent dye that is commonly added to agarose gels. This dye intercalates between the bases of DNA, allowing DNA fragments to be located in the gel under UV light and photographed. The intensity of the band reflects the concentration of molecules that size, although there are upper and lower limits to the sensitivity of dyes. Because of its interaction with DNA, ethidium bromide is a powerful mutagen and will interact with the DNA in your body just as it does with any DNA on a gel. You should always handle all gels and gel equipment with gloves. Agarose gels with Ethidium Bromide must be disposed as hazardous waste in the labelled container in the fume hood.

Today you will run your M13KO7 digested samples on an agarose gel, cut the linearized backbone out of your gel and then purify the DNA from the agarose. Next time you will mix the backbone and insert in a ligation reaction.

Part 1: Running your gel

• Add 2.5 µl loading dye to M13KO7 samples from last time. Loading dye contains xylene cyanol as a tracking dye to follow the progress of the electrophoresis (so you don’t run the smallest fragments off the end of your gel!) as well as glycerol to help the samples sink into the well.
• Flick the eppendorf tubes to mix the contents then quick spin them in the microfuge to bring the contents of the tubes to the bottom.
• Load the gel in the order shown in the table below. One group will load in wells 1 through 3, another group will load in wells 5 through 7. To load your samples, draw 25 µl into the tip of your P200. Lower the tip below the surface of the buffer and directly over the well. You risk puncturing the bottom of the well if you lower the tip too far into the well itself (puncturing well = bad!). Expel your sample into the well. Do not release the pipet plunger until after you have removed the tip from the gel box (or you’ll draw your sample back into the tip!).
• Once all the samples have been loaded, attach the gel box to the power supply and run the gel at 125V for no more than 45 minutes.
• You will be shown how to photograph your gel and excise the relevant bands of DNA.

Loading a gel. (Figure by MIT OpenCourseWare.)

Part 2: Isolating/Purifying DNA

To purify your DNA from the agarose, you will use a kit sold by Qiagen. The reagents have uninformative names and their contents are proprietary. The Agarose Purification kit requires binding the DNA to a spin-column with a silica-gel membrane, washing away salts and eluting the DNA from the membrane.

• Add 550 µl of QG to your slice of agarose.
• Incubate at 50°C for 10 minutes until the agarose is completely dissolved. Every few minutes, you can remove your tube from the 50°C heat to flick the contents. This will help dissolve the agarose.
• Add 125 µl of isopropanol to your eppendorf tube.
• Get a QIAquick spin column (purple) with collection tube (clear) from the teaching faculty. Label the spin column (not the collection tube!) then pipet the dissolved agarose mixture in the top of the column. Microfuge the column in the collection tube for 60 seconds. The maximum capacity of the QIAquick columns is 800 µl! If you have more than 800 µl in your mixture, you will need to repeat this step.
• Discard the flow-through in the sink and replace the spin-column in its collection tube. Add 750 µl of PE to the top of the column and spin as before.
• Discard the flow-through in the sink and replace the spin-column in the collection tube. Add nothing to the top but spin for 60 seconds more to dry the membrane.
• Trim the cap off a new eppendorf tube and label the side with your team color and the date. Place the spin-column in the trimmed eppendorf tube and add 30 µl of EB to the center of the membrane.
• Allow the column to sit at room temperature for one minute and then spin as before. The material that collects in the bottom of the eppendorf tube is your purified plasmid backbone, ready to be ligated. Give it to the teaching faculty who will store it with your insert until next time.

Part 3: Titering Phage

One technique you will see several times this term is plating for plaques. The idea of this technique is simple. Since phage infection slows down the growth of bacteria, any phage-infected cell will grow less quickly than an uninfected one, giving rise to a zone that is more clear on a lawn of fully grown cells. This zone is called a plaque and by counting the number of plaques formed, it is possible to measure the number of infective phage in the sample you are testing. The number of infected phages is measured as PFUs, which is “plaque forming units per ml.”

Plaques formed by bacteriophage upon infection of susceptible bacteria. Source: Assorted Views of Bacteriophage Plaques. © Quiroz. Licensed for use, ASM MicrobeLibrary .

• Start by placing 6 LB plates in the 37° incubator to prewarm them. If there is any condensation on the surface of the plates, then you can leave the lids slightly ajar to dry the plate surface.
• Aliquot 200 µl of bacteria into 6 small, sterile test tubes. The bacteria you are using are the strain ER2267 since this strain has a selectible F’. Label the tops of each tube with a colored sticker and one of the following: none, none, 10 -4 E4, 10 -6 E4, 10 -4 K07, 10 -6 K07.
• The teaching faculty have two phage stocks for you to compare, an M13KO7 phage stock and a stock of another M13 phage called E4. You will need to serially dilute each stock, making stepwise 1/100 dilutions in eppendorf tubes. For example, add 10 µl of a phage stock to 990 µl sterile water for a 10 -2 dilution, then repeat, using 10 µl of the 10 -2 dilution into 990 µl sterile water to make a 10 -4 dilution. Vortex the dilutions before removing any liquid and change pipet tips to prepare each new dilution. Continue serially diluting the phage to final concentrations that are 10 -4 th and 10 -6 th as concentrated as the starting stock.
• Mix 10 µl of one of the 10 -4 dilutions into a tube with bacteria.
• Mix 10 µl of one of the 10 -6 dilution into another tube with bacteria.
• Repeat with the dilutions of the other phage stock.
• One of the teaching faculty will show you how to mix 3 ml of top agar into one of the uninfected samples you have prepared and how to pour the molten mix onto the surface of a prewarmed LB plate.
• You and your partner should add top agar to the other uninfected sample and the four phage infected ones.

Allow the top agar to solidify by leaving the plates on the bench at least 5 minutes then stack them and wrap them with your colored tape and finally move them to the 37° incubator to grow overnight. One of the teaching faculty will remove them from the incubator tomorrow and store them for you until next time.

For Next Time

• Take the log10 of the length of each molecular weight marker you can identify on your agarose gel photograph. Graph the log 10 of their length on the y-axis versus the distance they migrated from the well on the x-axis, measured in mm using a ruler and the picture of your agarose gel. An example of such a graph is found in the introduction to today’s experiment. Use the equation of the line from your graph to determine the size of your M13KO7 backbone (use the band in the lane in which you loaded the cut DNA). How does this measurement compare with the predicted size?
• How many plaques do you expect if you plated 10 µl of a 10 -8 dilution of phage, if the titer of phage was 10 12 th plaque forming units/ml? How many plaques would you expect if you tested the phage stock on strain DH5?
• Tucker, Jonathan B., and Raymond A. Zilinskas. “The Promise and Perils of Synthetic Biology.” (PDF) New Atlantis, Spring 2006.
• Stone, Marcia. “Life Redesigned to Suit the Engineering Crowd.” (PDF) Microbe, Fall 2006.
• Marguet, P., et al. “Biology by Design: Reduction and Synthesis of Cellular Components and Behaviour.” Journal of the Royal Society Interface 4 no. 15 (August 22, 2007): 607-623. ( PDF )

Reagents list

• 0.25% xylene cyanol
• 30% glycerol
• 1% agarose gel in 1X TAE
• 40 mM Tris-acetate
• 1 kb Marker
• 50 mM Tris-HCl
• 10 mM MgCl2
• 25 μg/ml BSA

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