Agarose gel electrophoresis
10 questions in this handout
Electrophoresis is a general term to describe a number of methods that rely on the movement and separation of charged macromolecules (proteins and nucleic acids) in an ionic solution in response to an electric field. The basic components of electrophoresis include the
- Electric current as driving force
- Sample to be separated
- Support matrix (e.g., agarose gel)
- Buffer (e.g., Tris EDTA, ethylene diamine tetraacetic acid)
- Staining system (e.g., methylene blue and EtBr)
Nucleic acids are negatively charged, thus both RNA and DNA will migrate through a gel towards the positive (anode) electrode. The gel acts as a sieve, impeding the progress of larger molecules through the matrix. Thus, smaller molecules migrate faster than larger molecules through the gel. The rates by which the molecules travel are inversely related to their size.
Question 1: In running DNA, we say “Run to red!” Which end should the gel’s wells be located, at the anode or at the cathode?
To accurately determine molecular weights of unknown fragments, all samples should be run in parallel with a known standard or ladder. Agarose (a natural polymer) is used for relatively large DNA fragments, but does not resolve molecules that are similar in size; polyacrylamide (a synthetic polymer, and a neurotoxin, to boot!) is used for small DNA fragments because of it’s higher resolving power. For more information about electorphoresis, please see the numerous Wikipedia entries on the subject (wikipedia Electrophoresis; wikipedia Gel electrophoresis; wikipedia DNA electrophoresis).
If you have never run a gel before, then try this Virtual Lab exercise. It is kinda cheesy, but gives a good overview of DNA electrophoresis and analysis.
We will routinely use agarose gels to help us visualize runs of PCR products. In particular, we will run many RAPD and VNTR or SSR samples this semester. However, agarose is not appropriate for quantifying differences among the strains of flies for SSR because agarose is only capable of discriminating among DNA fragments that differ by several hundred bases. By definition, we expect microsatellites to vary among individuals by a few bases at most. However, agarose gels are easy to make and run and very appropriate for checking to make sure that our DNA extraction and PCR protocol are capable of providing us with product.
In the case of SSR gels, and assuming we have product, we will use agarose gels as preparative gels. We can run the gels, then cut the bands from the gels and save the product because, once we have established that we have product, we will proceed next time to denaturing polyacrylamide gels. Polyacrylamide gels are capable of discriminating among DNA fragments that may differ by no more than one base.
Components of electrophoresis system
Question 2: On the exam, you should be able to draw and correctly label the components of an electrophoresis apparatus.
Figure 1. Thermo OWL B1 mini gel electrophoresis system in action (agarose gel).
Gel box and buffer tanks:
For running nucleic acid fragments, a horizontal or “submarine” gel box is used. Gel boxes are made of high impact resistant plastic. At either end you will see a single thin wire, the electrode, which runs to the bottom and across each buffer tank. The electrodes are connected to plugs that are used to connect the gel box to the power supply leads. Note that the box has a buffer tank at both ends of a raised platform. The gel is covered with the running buffer and rests on that platform.
Power supply:
The power supplies we use deliver DC — direct current — via the connected leads to the gel box. You can set the power supply to deliver constant volts (V) or constant current (I) in amps. Following Ohm’s law, V = IR, where R is the resistance, the “opposition” to the flow of current. The concentration of agarose and buffer, plus the gel thickness all determine the resistance — in principle, the higher the resistance, the greater separation of DNA fragments.
Question 3. During a gel electrophoresis run, you should monitor the current to confirm the buffer is in working order. If the volts are equal to 140 and the current reads 100 milliAmps, what is the resistance?
In general, the higher the volts the more “force” is generated and the faster the nucleic acid fragments will move in the gel, at the expense of release of lots of heat, which may melt the gel or cause other and potentially more hazardous consequences. As long as the running buffer is fresh, good connectivity will be achieved between the anode and cathode. During the run, monitor milliAmps: if the milliAmps drop, then the running buffer has lost much of the ions needed to maintain the current flow.
Buffers:
The running buffer, the volume of liquid in the electrophoresis box, is the same buffer used to make the gel, and at the same concentration (e.g., 1X TAE). Running buffer is either TAE (tris-acetate-EDTA; Wikipedia), or TBE (tris-borate-EDTA; Wikipedia), buffers are used to maintain pH and to provide ion support conductivity.
Question 4. What is significant about “ion support conductivity” in electrophoresis?
Tris (Wikipedia) keeps DNA deprotonated and soluble in water. EDTA (Wikipedia EDTA) is a chelating agent – it forms complexes with many divalent metal ions that that are required co-factors for enzymes. Thus, EDTA is also important in storage of DNA or other nucleic acids.
The electrophoresis part is just electrolysis. What is the nature of the bubbles of gas in Figure 2? In Figure 3?
Figure 2. Bubbles at “red” buffer end.
Figure 3. Bubbles at “black” buffer end.
Question 5. Why are there twice as many bubbles at the “black” electrode than at the “red” electrode during agarose gel electrophoresis?
Question 6: So, under these conditions, is DNA an acid or a base or neither?
Question 7. With respect to “accepting” or “releasing” H+ (hydrogen ions), what is an acid? What is a base.
Question 8: During an electrophoresis run, heat is inevitably generated. It is known that Tris is a poor buffer as temperature increases. Why is this an important consideration for running electrophoresis of DNA for more than 2 hours?
Figure 4. Loading DNA samples into the gel.
Loading buffer: a buffer that contains 25% v/v glycerol or sucrose or Ficoll plus a tracking dye (e.g., bromophenol blue). The tracking dye allows progress of the electrophoresis run to be monitored without the need for visualizing the bands.
Question 9. What is the purpose of the glycerol (or sucrose or Ficoll)?
We need to make visual the biological molecules moving in the gel. We “stain” the molecules. Many chemical agents have been found or developed to make DNA visible, one of the oldest known is ethidium bromide. EtBr: Ethidium bromide (Wikipedia EtBr) is useful for staining DNA and RNA, but is highly toxic and a mutagen. Under no circumstances should EtBr be used without safety precautions (glasses, gloves), and disposal must be conducted appropriately (see instructor and MSDS). EtBr binds to DNA bases, producing an orange color visible under UV illumination. EtBr intercalates (inserts) between the base pairs of double-stranded DNA. Under proper conditions, approximately one EtBr molecule is intercalated per 3 base pairs, regardless of the DNA sequence. EtBR may be added to the gel before loading the sampe DNA, which permits visual monitoring of the band progress during electrophoresis, or it may be added after the gel has been run, thus eliminating impact on mobility of DNA that EtBr will likely have.
Some researchers prefer to add EtBr to the buffer or gel so that the bands can be viewed in real time; others prefer to add EtBr to the gel after a run has been completed. There are pros and cons to either choice. We will stain the gel afterwards; this cuts down on EtBr waste.
Question 10. What is the proper procedure for disposing of etbr waste after staining a gel?
A relatively nontoxic alternative to EtBr is methylene blue: also a useful, and nontoxic stain for DNA and RNA. Binding of the dye to DNA seems to be ionic, to the phosphate groups. Visualization of bands is also conducted under UV light. It is less sensitive then EtBr. A solution diluted to 0.02 – 0.04% in water is typical. If approximately 50 ng of DNA (or RNA) are used per lane and a procedure of staining for 1 hour is employed, desirable results are achieved. We do not use this dye because it requires significantly more DNA than we are likely to have in each band.
References
Brody, JR & SE Kern (2004) History and principles of conductive media for standard DNA electrophoresis. Analytical Biochemistry 333:1-13
Sanderson, B. A., Araki, N., Lilley, J. L., Guerrero, G., & Lewis, L. K. (2014). Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis. Analytical Biochemistry, 454:44–52.
Westermeier, R 2005 Electrophoresis in practice, 4th ed. Wiley