Title of Experiment 9: Measurement of Nucleic Acid Solutions
Objectives:
1. To study the principle of gel electrophoresis. 2. To use gel electrophoresis method to measure the nucleic acid solutions. 3. To learn the technique of nuclei acid measurement by using gel electrophoresis.
Introduction:
Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. The components of gel electrophoresis system is power supply and a chamber, agarose gel which is a porous material that allows molecules migrate through, buffer with a mixture of water and ions, and gel casting tray and comb.
Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. Nucleic acids are composed of chains of nucleotides. The ‘backbone’ of the nucleic acid structure is a repeating chain of phosphate groups and pentose sugars. At certain pH values, the oxygen atoms in the phosphate groups ionize, giving the molecule an overall negative charge. If exposed to an electric field, these molecules are attracted to the positive terminal.
During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical current. Under an electrical field, DNA will move from the negative electrode (black) to the positive electrode (red). Several factors influence speed of the DNA movement, including; the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules. This is because smaller fragments of DNA molecules can penetrate further through the gel than larger fragments in the same time. This means that bands of smaller DNA fragments will be found closer to the positive terminal, while larger fragments will be found closer to the loading wells.
DNA itself is not visible within an agarose gel. Therefore, the DNA is visualized by the use of a dye that binds to DNA, ethidium bromide. It binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light. DNA concentration can be compared between in the intensity of the stained bands with the marker bands of known mass.

Apparatus and Materials:
Electrophoresis chamber, power supply, comb, microwave oven, conical flask, casting tray, staining tray, gloves, P-10 micropipette and tips, UV transilluminator, parafilm, agarose gel powder, Tris-borate-EDTA (TBE) buffer, ethidium bromide, PUC19 DNA, λHindIII marker, and bromphenol blue loading dye.

Procedures:
Agarose gel electrophoresis of DNA A. Casting the gel

B. Preparing samples

C. Loading and Running the gel

D. Staining the DNA in the gel with ethidium bromide

E. Photography

Results:
Figure [ 2 ] shows the gel electrophoresis results of λHindIII marker in the first track which compared its size with Figure 1 and pUC19 DNA in the second to the fifth tracks with fragment label (2686 bp).

Figure [ 1 ] shows the sizing and approximate quantification of Lambda DNA/HindIII Marker, 2 (Lambda HindIII Marker).
2686 bp

There are total of five wells filled with DNA. The first well is λHindIII marker where as the second to the fifth wells were filled with pUC19 DNA. 5µl of each DNA was mixed with 1µl of loading dye so that the DNA samples can be tracked.
The actual base pairs count for pUC19 DNA is 2686 bp according to Thermo Fisher Scientific Inc. The bands of pUC19 DNA stop migrated around 2322 bp and 2027 bp of the λHindIII marker.
Noted that the 23130 bp of the marker did not appeared in Figure 2 may be due to its larger weight, which need a longer time to migrate through the gel.

Discussion:
In the experiment, the DNA loading dye bromophenol blue 6x was used contains something dense 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 proceed. The loading dye migrates at approximately 300 bp on a standard 1% TBE agarose gel (Gel Loading Dye, Blue).
From the result of agarose gel electrophoresis experiment, Figure 2 shown the bands of six DNA tracks which contain of λHindIII marker and pUC19 DNA. Different bands indicated different fragment sizes when the smaller it is, the faster it travels and the lower position it is in the image. Besides, the different intensities indicated different concentrations of DNA where the brighter the loaded sample, the more DNA it contains. The DNA was made visible by staining with ethidium bromide and view under ultraviolet light (CASEY, 2006).
There are several factors that have effects on the mobility of DNA fragments in agarose gel electrophoresis. One of the factors is agarose concentration. Different sizes of DNA fragments dissolved in different concentrations of agarose gel. Higher concentrations of agarose gel facilitate the separation of smaller DNA and vice versa for lower concentration of agarose gel. Next, voltage applied also affects the mobility of DNA. As the voltage applied to the agarose gel increase, the larger fragments of DNA migrate proportionally faster. In this experiment, the electrophoresis buffer used is TBE where it is also one of the factors. DNA fragments will migrate at different rates in different buffers due to differences in ionic strength. Buffers not only establish a pH, but also provide ions to support conductivity. Moreover, ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in the agarose gel as shown in the result. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. The binding of ethidium bromide to DNA may alter its mass and rigidity, and also the mobility. The circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass (Rbowen, 2000).
Although ethidium bromide is a most commonly used fluorescent dye for DNA and RNA detection in gels, it is a very powerful mutagen and should be handled as a hazardous and toxic chemical. It also must be handled with extremely caution and decontaminated prior to disposal. Conversely, another staining product, SYBR Safe DNA gel stain is a less hazardous alternative to ethidium bromide that can be used with either blue-light or ultraviolet light. It is a highly sensitive stain for visualization of DNA in the agarose gel (SYBR13). Furthermore, always wear protective glove when dealing with ethidium bromide and DNA. Protective eyewear is necessarily when observing DNA on a transilluminator to prevent damage to the eyes from ultraviolet light. Besides, be sure that the door of the darkroom is closed properly before turning on the transilluminator so that a clearer photo can be taken. The DNA and loading dye were mixed on the parafilm to avoid any lost of chemicals by transferring to other surface.
Conclusion:
Agarose gel electrophoresis is a very useful method to measure nucleic acid depending on their bands and marker. From the experiment, we learnt that the base pairs of restricted DNAs by enzyme can be compared with its ladder DNA by using agarose gel electrophoresis method. The result of this experiment indicated that pUC19 DNA can be measured with λHindIII marker.

References:

Anon., n.d. Gel Loading Dye, Blue (6X). [Online]
Available at: https://www.neb.com/products/b7021-gel-loading-dye-blue-6x
[Accessed 30 March 2013].
Anon., n.d. Lambda DNA/HindIII Marker, Ready-to-Use. [Online]
Available at: http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/lambda-dna-hindiii-marker-ready-to-use/
[Accessed 02 April 2013].
Anon., n.d. pUC18, pUC19 DNA. [Online]
Available at: http://www.thermoscientificbio.com/molecular-cloning/puc18-puc19-dna/
[Accessed 02 April 2013].
Anon., n.d. SYBR® Safe DNA Gel Stain. [Online]
Available at: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/DNA-Stains/SYBR-Safe.html
[Accessed 31 March 2013].
CASEY, S., 2006. Comparing DNA. [Online]
Available at: http://www.learnnc.org/lp/editions/american-chestnut/6972
[Accessed 30 March 2013].
Rbowen, 2000. Agarose Gel Electrophoresis of DNA. [Online]
Available at: http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
[Accessed 31 March 2013].