Agarose gel electrophoresis

Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. It operates by a mechanism similar to sifting molecules through a sieve; an electric field is used to drag negatively charged DNA molecules through a gel matrix, and the shorter DNA molecules move faster than the longer ones since they are able to slip through the gel more easily. Proteins can also be separated due to different charges and sizes. It can be used for the separation of DNA fragments of 50bp up to several megabases. Large DNA molecules are only able to move end on in a process called "reptation" and are more difficult to separate. In general the lower the concentration of agarose, the larger is the ideal size of a molecule to be resolved up to 750Kb. The disadvantage of lower concentrations is the long run times (sometimes days) and the problem of handling the fragile gel. The rate of migration is affected by a number of factors. The concentration of agarose is one that has been mentioned. The conformation of DNA is also a factor and is demonstrated by the three forms of a plasmid: superhelical, nicked, and linear. Each form runs differently, the superhelical the fastest and the linear form the slowest. The presence of ethidium bromide (EtBr) in the gel causes DNA to run slower due to EtBr's ablilty to intercalate and uncoil DNA. The voltage is also a factor in migration and can only be so high before a decrease in resolution (~5-8 V/cm). Loading buffers are added with the DNA in order to visualize it and sediment it in the gell well. Negatively charged indicators keep track of the position of the DNA. Bromphenol Blue and Xylene cyanol FF are used and run at about 300bp and 4kb respectively. There are a number of buffers used for agarose electrophoresis, but I will only mention two: tris acetate EDTA (TAE), and sodium boride (SB). TAE has the lowest buffering capacity but provides the best resolution. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5kb but it has the highest buffering capacity allowing voltages up to 350V⁵,⁶.

Material

For an agarose gel electrophoresis, several items are needed:

• The DNA that is to be separated.
• A DNA ladder, a mixture of DNA fragments (usually 10-20) of known size. The size of the DNA strands that are separated is determined by comparison of their relative position to that of the DNA strands of the DNA ladder. There are several DNA ladder mixes commercially available.
• Buffer, usually TBE or TAE 0.5x, pH 8.0
• Agarose
• Ethidium bromide (5.25 mg/ml in H₂O)
• Nitrile gloves
• A color marker 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" (usually cut from a sheet of teflon)

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 minimum while length and breadth are varied as needed), and whether the gel is prepared horizontally or vertically (the vast majority of agarose gels used in modern molecular biology are prepared and run horizontally).

1. Make a 1% agarose solution in 0.5x TBE. If you analyze small DNA strands, go up to 2%. Use 15-70 ml, depending on the size of the gel.
2. Boil solution, preferably in a microwave oven.
3. Let the solution cool down to about 60°C at room temperature. Stir the solution while cooling.
4. Add 1 ml ethidium bromide on each 10 ml gel solution. Wear gloves from here on, ethidium bromide is a mutagen (nitrile gloves recommended) ! Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
5. Stir the solution to disperse the ethidium bromide, then fill it into the gel rack.
6. Insert the comb at one side of the gel, about 5-10 mm from the border of the gel.
7. When the gel has cooled down and become solid, remove the comb. The holes that remain in the gel are the slots.
8. Put the gel, together with the rack, into a chamber with 0.5x TBE. Make sure the gel is completely covered with TBE, and that the slots are at the electrode that will have the negative current.
9. Add the color marker to the The DNA ladder is usually already stained.

for more information on ethidium bromide safety see references ¹,²,³

for information on alternatives to ethidium bromide see references ²,⁴

Procedure

After the gel has been prepared, use a micropipette to inject about 25 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100V 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 know possible to visualize the DNA (stained with ethidium bromide) with UV light.

Steps:

1. The agarose gel with three slots (S).
2. Injection of DNA ladder 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. The DNA is spread over the whole gel. The electrophoresis process is finished.

Illuminate the gel with an ultraviolet lamp (usually by placing it on a light box) to view the DNA bands - ethidium bromide fluoresces pink in the presence of DNA. Wear protective glasses! The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.

References

Note 1: States, Kelly M. (2003). Ethidium Bromide in The Waste-Paper:The Hazardous Waste Disposal Monthly Update (http://web.princeton.edu/sites/ehs/chemwaste/WastePaper/0302.htm). Retrieved 2005-01-31. Note 2: Office of Biological Safety, Univ. of Wisconsin (Madison) (2003). Ethidium Bromide: Alternatives and Safe Handling in BioSide Lines:The Newsletter of the UW Office of Biological Safety (http://www2.fpm.wisc.edu/biosafety/bioside_lines/bioside_lines_04_2003.htm). Retrieved 2005-01-31. Note 3: Environmental Health and Safety at The Scripps Research Intititute (1999). WILL YOUR GLOVES PROTECT YOU? in Environmental Health & Safety:Second Quarter 1999 (http://www.scripps.edu/researchservices/ehs/News/safetygram/sg1999/sg1999b.html) Retrieved 2005-01-31. Note 4: Madden, Dean (2004 [last modified]). Safer stains for DNA (http://www.bioscience-explained.org/EN1.2/schollar.html). Retrieved 2005-01-31. Note 5: Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Note 6: 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] (http://www.biotechniques.com/default.asp?page=article_archive&subsection=article_display&id=101200415&prevpage=article_archive)

See also

• SDS-polyacrylamide gel electrophoresis
• Southern blot
• Northern blot
• PCR
• Restriction endonucleasede:Agarose-Gelelektrophorese

ja:アガロースゲル電気泳動