Agarose Gel Electrophoresis of DNA - Lab Report Example

? Agarose Gel Electrophoresis of DNA DNA is the double stranded helical structure which carries the genetic information. The study of the DNA molecule will give us the details about life. DNA molecules can be extracted from the cell using the extraction techniques and they are then quantified using the agarose gel electrophoresis. (Westermeier 2006). Agarose gel electrophoresis is the most common and easiest technique to separate the DNA fragments. Agarose is a polymer that forms helical linking strands between the molecules and they are held together by hydrogen bonds. Based on the concentration of the agarose in the gel, the pore size also varies. The DNA fragment is separated in the Agarose based on the pore size (that is the concentration of the agarose), the voltage applied, the molecular size of the DNA molecule and the conformation of the DNA. The smaller molecules a move downwards in the gel faster than the larger molecules and the separation occurs based on the size of the DNA. If high voltage is applied, then the migration will be faster but the separation of the DNA fragments will not be clear. (Westermeier 2006). So, 110 volts for 30- 45 minutes is usually applied for the separation of the DNA fragments in the gel. The agarose gel electrophoresis of the DNA molecule showed distinct bands in the lanes 4,5 and 6 indicating that only a single DNA is present in the sample. Introduction: Doeoxy ribo nucleic acid (DNA) is a polymer consisting of base, sugar and a phosphate bond. The sugar is always deoxy- ribose in case of DNA and the base will be a purine or pyrimidine molecule: Adenine, Guanine, Cytosine or Thymine. Phosphate molecule connects the two nucleosides. DNA is a covalently linked structure which is helical in shape. DNA is a double helical structure held together by the hydrogen bonds. DNA carries the genetic information. The analysis to the DNA thus will help us to understand all the basics of life, so separation and purification of DNA is required for further studies. One of the most common and easiest methods to separate the DNA and RNA fragments is by Electrophoresis. DNA electrophoresis started around 1964. This separation is based on the size. In this method, agarose gel acts as a sieve and separates the DNA fragments based on their molecular weight under the influence of electric field. Since DNA are acidic in nature, they migrate towards the positive pole when exposed to an electric field. The pore sixe of agarose sieve is determined by the concentration of agarose. Agarose gels are made with a concentration varying from 0.7- 1.5%. DNA in a neutral solution is negatively charged. (Williamson and Campbell 1997). So if an electric field is applied to the DNA, it will move towards the anode pole from the cathode pole. Based on the fragment size, the rate of migration will be inversely proportional to the fragment size. The smaller fragments will move faster than the larger ones and the distance moved by the fragments are measured by using the molecular marker. Molecular marker is the standard DNA fragment sizes which act as the standards to measure the molecular weight of the DNA fragments. Thus by providing constant voltage into the agarose gel, we are able to separate the DNA fragments based on their molecular weight. (Westermeier 2006). Agarose Gel Electrophoresis of DNA is a very simple and reproducible technique. The mobility of the DNA molecule in neutral solution was independent of the size of the fragment but varied with the ionic strength .The DNA fragments of up to 40 kilo base pair can be separated using agarose gel electrophoresis. (Williamson and Campbell 1997). The DNA fragments get separated based on molecular size, the current applied, concentration of Agarose and the conformation of the DNA. Agarose is a copolymer containing 1,3-linked ?-D-galactose and 1,4-linked 3,6- anhydro-?-L-galactose linked together by the junction zones and joined by the hydrogen bonds. (Stellwagen 2009). A standard ladder is used to identify the size of the fragments. In electrophoresis, the gel is casted in an appropriate size tray or mould. A comb is placed on the gel in the liquid stage and allowed to harden. After the gel has hardened, the comb is removed. The wells in the gel are used for loading the samples. The gel is then placed in the horizontal electrophoresis and covered by the buffer. The samples are then loaded onto the gel and current is applied for a particular time and the result is quantified. (Clark and Pazdernik 2012). The Bands on the gel are visualized using the intercalating fluorescent dye, Ethidium bromide. When the Agarose gel dyed with EtBr is visualized in the ultra violet rays (UV rays), we can observe the bands. The Ethidium Bromide is a florescent dye that gets illuminated under the UV light. When UV light of wavelengths 260, 300 and 360 nm are used, the DNA bands can be observed very efficiently almost to the concentration of 1 nanogram. Ethidium Bromide binds to the intercalating planar group between the base pairs of nucleic acid. This method is very efficient as it can detect the dsDNA concentration as low as 1 nanogram. (Robertson, Ross and Burgoyne 1990). Materials and Methods: Materials Required: 0.5g of Agarose. 50 ml of Distilled water. 1 x TBE buffer – it is prepared by mixing 89mM Tris base, 89mM Boric Acid and 2mM EDTA. The pH is adjusted to 8.0 Ethidium Bromide. ( 0.5 microgram / milliliter ). Gel casting tray, comb. Electrophoresis unit. Power supply. Bromophenol Blue – staining dye. Method: Preparation of Agarose Gel: 1. 1% Agarose gel was prepared by mixing 0.5 gram of Agarose in 50ml of water and boiling it until it completely dissolves in the buffer. The heating can be done for approximately 1 minute and 20 seconds. 2. Gel plate was taped on three sides and the gel was poured into the gel casting tray. 3. The comb is inserted into the gel. The gel is then allowed to solidify for 30 minutes at Room temperature. Sample preparation: 8 µl of the sample DNA was mixed with 2 µl of Bromophenol Blue. A total of 10 µl is required for each well. Electrophoresis: 1. The Electrophoresis tank was kept ready and the 1x TBE buffer was poured on to the Unit. The anode and cathode units are now immersed in the buffer. 2. The gel was then placed on the tray and the remaining buffer was added, till the gel is almost immersed in the buffer. 3. 10 µl of the sample DNA was loaded along with the Bromophenol blue. 4. The Electrophoresis set up was run at 110 V for 50 minutes. After that the power is turned off and the gel was removed from the tray and kept in the plastic container. Examining the Gel: 1. The gel is stained with Ethidium Bromide at a concentration of 0.5µg/ ml. The gel is submerged in the solution for 10 minutes. This post-Electrophoresis method reduces the wastage of Ethidium bromide when compared to adding Ethidium Bromide in the gel when solidifying. 2. The gel was then de-stained in distilled water for 5 minutes. 3. The gel was kept under UV transilluminator and the DNA bands are visualized. The orangish pink bands were observed and the results were noted. Result: Figure 1: Agarose Gel Electrophoresis of DNA Discussion: The DNA is observed using the agarose gel electrophoresis. Here the DNA is seen as distinct bands. Lanes 4, 5 and 6 shows intense DNA bands indicating that the concentration is high. Thus by using agarose gel electrophoresis DNA can be observed and eluted. References: Clark, DP and Pazdernik, NJ., 2012. Molecular Biology, Elsevier. Robertson, JR., Ross, AM and Burgoyne, L., 1990. DNA In Forensic Science: Theory, Techniques And Applications, Routledge. Stellwagen, NC., 2009. Electrophoresis of DNA in Agarose Gels, Polyacrylamide gels and in Free solution, Electrophoresis, Vol. 30, No.1, pp. S188–S195. Williamson, JH and Campbell, AM., 1997. DNA Technology in the Classroom: A Circular Map of a Bacterial Plasmid, The American Biology Teacher, Vol. 59, No. 3, pp. 164-70. Westermeier, R., 2006, Electrophoresis in practice, John Wiley and Sons. Read More