Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. The DNA size marker is a commercial 1kB ladder. The position of the wells and direction of DNA migration is noted.

Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate further than longer ones.^[1] Image File history File links Size of this preview: 347 × 598 pixel Image in higher resolution (828 × 1428 pixel, file size: 141 KB, MIME type: image/png) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis... Image File history File links Size of this preview: 347 × 598 pixel Image in higher resolution (828 × 1428 pixel, file size: 141 KB, MIME type: image/png) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis... Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules through an electric charge. ... Biochemistry is the study of the chemical processes in living organisms. ... Molecular biology is the study of biology at a molecular level. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... In physics, the space surrounding an electric charge or in the presence of a time-varying magnetic field has a property called an electric field. ... It has been suggested that Electrophoretic mobility be merged into this article or section. ...

Applications

• Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.
• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
• Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ... Wikipedia does not yet have an article with this exact name. ... In medicine and (clinical) genetics preimplantation genetic diagnosis (PGD) is a method to test oocytes prior to fertilization or embryos before they are implanted in the uterus. ... Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... The northern blot is a technique used in molecular biology research to study gene expression. ...

The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms. Ur mama

Factors affecting migration

The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of the DNA molecule is also a factor. To avoid this problem linear molecules are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. Conformation generally means structural arrangement. ... A restriction digest is a molecular biology procedure used to prepare DNA for analysis or other processing. ... Wikipedia does not yet have an article with this exact name. ...

Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm). ^{[citation needed]}

Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid. Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ...

Visualisation: EtBr and dyes

The most common dye used for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible. ^{[citation needed]}. Alternatives to ethidium bromide are available. R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic...

Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Xylene cyanol and Bromophenol blue are typically used. They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp.^{[citation needed]} Xylene cyanol can be used as a color marker to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis; in agarose gels, it typically migrates at about the same rate as a 4000 base pair DNA fragment. ... Bromophenol Blue Bromophenol blue, Tetrabromophenolsulfonphthalein, is an acid-base indicator whose useful range as an indicator lies between pH 3. ... Cresol Red (full name: o-Cresolsulfonephthalein) is a triarylmethane dye frequently used for monitoring the pH in aquaria. ... Orange G Orange G, Acid Orange 10, or C.I. 16230, is a synthetic azo dye used in histology in many staining formulations. ...

Resolution limits

Gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). However, it is normally used in a range of 100 bp to 20 kbp. Typical run times are about an hour. Base pairs, of a DNA molecule. ...

Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules are only able to move end-on in a process called "reptation" and are more difficult to separate. In general higher concentrations of agarose are better for larger molecules; it will exaggerate the distances between bands. The disadvantage of higher concentrations is the long run times (sometimes days). Instead these gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis. This article does not cite any references or sources. ... Pulsed Field Gel Electrophoresis (commonly abbreviated as PFGE) is a labor-intensive method for genetic fingerprinting. ...

Agarose

Agarose is purified from agar, a gelatinous substance isolated from seaweed or red algae. Different purities of agarose are commercially available as are agaroses with different melting properties. High purity low melt agarose is often used if the DNA is to be extracted from the gel. This does not adequately cite its references or sources. ... Ascophyllum nodosum exposed to the sun in Nova Scotia, Canada Dead Mans Fingers (Codium fragile) off Massachusetts coast For the band, see; Seaweed (band) For the rock musician, see; Seaweed (musician) Seaweeds are any of a large number of marine benthic algae. ... Possible classes Florideophyceae Bangiophyceae Cyanidiophyceae The red algae (Rhodophyta, IPA: , from Greek: (rhodon) = rose + (phyton) = plant, thus red plant) are a large group, about 5000 - 6000 species [1] of mostly multicellular, marine algae, including many notable seaweeds. ...

Buffers

There are a number of buffers used for agarose electrophoresis. The most common being: tris acetate EDTA (TAE), Tris/Borate/EDTA (TBE) and Sodium borate (SB). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).^[2] EDTA is a widely-used acronym for the chemical compound ethylenediamine tetraacetic acid (and many other names, see table). ... TBE buffer is widely used for the electrophoresis of nucleic acids and has a higher buffer capacity than TAE. It can be used for DNA and RNA polyacrylamide and agarose gel electrophoresis. ... Borax, (Na2B4O7·10H2O, sodium borate or sodium tetraborate) is an important boron compound. ...

Analysis

After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA bands. The ethidium bromide fluoresces reddish-orange in the presence of DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white (see figures). For other uses, see Ultraviolet (disambiguation). ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ...

Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ. ImageJ is a public domain, Java-based image processing program developed at the National Institutes of Health. ...

1	2	3
An Agarose 'slab' gel prior to UV illumination, behind a perspex UV shield. Only the marker dyes can be seen	The gel with UV illumination, the ethidium bromide stained DNA glows pink	Digital photo of the gel. Lane 1. Commercial DNA Markers (1kbplus), Lane 2. empty, Lane 3. a PCR product of just over 500 bases, Lane 4. Restriction digest showing the a similar fragment cut from a 4.5 kb plasmid vector

Example of DNA agarose gel electrophoresis. ... Example of DNA agarose gel electrophoresis. ... Image File history File linksMetadata No higher resolution available. ... Image File history File linksMetadata No higher resolution available. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... Image File history File linksMetadata No higher resolution available. ... Image File history File linksMetadata No higher resolution available. ... Wikipedia does not yet have an article with this exact name. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ... Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. ...

Typical materials and method

Materials

For an agarose gel electrophoresis, several items are needed:^{[citation needed]}

• DNA fragments to separate; typically 10-30 μl/sample
• DNA size markers.

A mixture of DNA fragments (usually 10-20) of known size. DNA size markers may be purchased from a commercial source or prepared manually.

• Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0
• Agarose
• Ethidium bromide stock (5.25 mg/ml in H₂O).

Alternative dyes may be used, such as SYBR Green.

• Nitrile rubber gloves

Latex gloves do not protect well from ethidium bromide

• A color marker dye containing a low molecular weight dye such as "bromophenol blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA solution more dense so it will sink into the wells of the gel).
• A gel rack
• A "comb"
• Power Supply
• UV lamp or UV lightbox or other method to visualize DNA in the gel

Acids and bases: Acid-base reaction theories pH Self-ionization of water Buffer solutions Systematic naming Electrochemistry Acid-base extraction Acids: Strong acids Weak acids Superacids Lewis acids Mineral acids Organic acids Bases: Strong bases Weak bases Superbases Lewis bases Organic bases edit Buffer solutions are solutions which resist change... TBE buffer is widely used for the electrophoresis of nucleic acids and has a higher buffer capacity than TAE. It can be used for DNA and RNA polyacrylamide and agarose gel electrophoresis. ... An agarose is a polysaccharide polymer material, generally extracted from seaweed. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... Chemical Structure of SYBR Green I Spectrogram of SYBR Green I SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. ... Nitrile rubber is a synthetic rubber co-polymer of acrylonitrile (ACN) and butadiene. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... The molecular mass of a substance (less accurately called molecular weight and abbreviated as MW) is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). ... Look up dye in Wiktionary, the free dictionary. ... Bromophenol Blue Bromophenol blue, Tetrabromophenolsulfonphthalein, is an acid-base indicator whose useful range as an indicator lies between pH 3. ...

Preparation

There are several methods for preparing agarose gels. A common example is shown here. Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a constant amount while length and breadth are varied as needed). The vast majority of agarose gels used in modern biochemistry and molecular biology are prepared and run horizontally.^{[citation needed]} Biochemistry is the study of the chemical processes in living organisms. ... Molecular biology is the study of biology at a molecular level. ...

1. Make a 2% agarose solution in 100ml TAE. A solution of up to 4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.8% is preferable. Use 15-100 mL, depending on the size of the gel.
2. Bring the solution to the boil to dissolve the agarose, preferably in a microwave oven.
3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or swirl the solution while cooling.

Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety see ethidium bromide It has been suggested that this article be split into articles entitled Microwave oven and Microwave heating. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... In biology, a mutagen (Latin, literally origin of change) is a physical or chemical agent that changes the genetic information (usually DNA) of an organism and thus increases the number of mutations above the natural background level. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic...

1. Add 5 µl ethidium bromide stock per 100 ml gel solution. Be very careful when handling the concentrated stock. Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
3. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
4. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots.
5. Put the gel, together with the rack, into a tank with 0.5x TBE. Ethidium bromide at the same concentration can be added to the buffer. Make sure the gel is completely covered with TBE, and that the slots are at the end electrode that will have the negative current.^{[citation needed]}

R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic...

Procedure

After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. It is now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light. ^{[citation needed]} For other uses, see Ultraviolet (disambiguation). ...

Steps: ^{[citation needed]}

1. The agarose gel with three slots/wells (S).
2. Injection of DNA ladder (molecular weight markers) into the first slot.
3. DNA ladder injected. Injection of samples into the second and third slot.
4. A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone.
5. Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.
6. Add the color marker dye to the DNA ladder.

Agarose gel with samples loaded in the slots, before the electrophoresis process

A pattern of DNA-bands under UV light

The molecular mass of a substance (less accurately called molecular weight and abbreviated as MW) is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). ... Diagram of a zinc anode in a galvanic cell. ... A phosphate, in inorganic chemistry, is a salt of phosphoric acid. ... Image File history File linksMetadata Download high-resolution version (1600x1200, 493 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis Metadata This file contains additional information, probably added from the digital camera or scanner used... Image File history File linksMetadata Download high-resolution version (1600x1200, 493 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis Metadata This file contains additional information, probably added from the digital camera or scanner used... Image File history File linksMetadata Download high-resolution version (1600x1200, 445 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis Metadata This file contains additional information, probably added from the digital camera or scanner used... Image File history File linksMetadata Download high-resolution version (1600x1200, 445 KB) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Agarose gel electrophoresis Metadata This file contains additional information, probably added from the digital camera or scanner used... Image File history File links No higher resolution available. ... Year 2007 (MMVII) is the current year, a common year starting on Monday of the Gregorian calendar and the AD/CE era in the. ... is the 172nd day of the year (173rd in leap years) in the Gregorian calendar. ...

References

1. ^ Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
2. ^ Brody JR, Calhoun ES, Gallmeier E, Creavalle TD, Kern SE (2004). Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques. 37:598-602. [1]

See also

• gel electrophoresis
• SDS-polyacrylamide gel electrophoresis
• Southern blot
• Northern blot
• PCR
• Restriction endonuclease

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules through an electric charge. ... Picture of an SDS-PAGE. The molecular marker is in the left lane SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... The northern blot is a technique used in molecular biology research to study gene expression. ... Wikipedia does not yet have an article with this exact name. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. ...

External links

• How to run a DNA or RNA gel

• Excellent animation of gel analysis of DNA restriction fragments

• Detailed description and movies of the preparation and uses of agarose gels

• Step by step photos of running a gel and extracting DNA