ELEKTROFORESIS

Electrophoresis is a technique or component separation of charged molecules based on differences in the level of migration in an electric field. The electric field was supplied to a medium containing the sample to be separated. This technique can be used by utilizing the existing electrical charge on the macromolecules, such as negative-charged DNA. If the negatively charged molecules that passed through a medium, then electrified pole to pole of an opposite charge so the molecules will move from the negative to the positive pole. The velocity of the molecule depends on the ratio of charge to its mass and depends also on the shape of the molecule.Molecules are moving at the speed of the electric field depends on the charge, shape and size. Thus can be used for the electrophoretic separation of macromolecules (such as proteins and nucleic acids). Terseparasi position on the molecule can be detected by gel staining or autoradiography, quantification with a densitometer or done. Electrophoresis for matrix macromolecules requires a buffer to prevent the diffusion of heat due to the onset of electrical current used.2.1.2 Types of Electrophoresis• paper electrophoresisElectrophoretic types of paper as the stationary phase and the charged particles are dissolved as the mobile phase, is particularly complex ions. This separation is the result of a concentration gradient along the separation system. The movement of particles in the paper depends on the charge or valence of the solute, the cross sectional area, the voltage used, the concentration of electrolyte, ionic strength, pH, viscosity, and solute adsorpsivitas.• gel electrophoresisElectrophoresis using the gel as the stationary phase to separate molecules. Originally performed by gel elektoforesis starch gel medium (as the stationary phase) to separate the larger biomolecules such as proteins. Gel electrophoresis and then grow by making the agarose gel and polyacrylamide as the media.o agarose gelsPhysically, the agarose seem like a very fine white powder. Agarose commercially sold contaminated with polysaccharides, salts and proteins. The extent of contamination in the gel and the ability to take DNA from the gel to be used as substrates in enzymatic reactions.o polyacrylamide gelThis gel formation is performed by heating, but by mixing a solution of acrylamide with ammonium persulfate and TEMED.• Electrophoresis of DNAIs a technique to separate DNA samples based on size (molecular weight) and physical structure of the molecule. Gel which is used among others agarose. Agarose gel electrophoresis can be done to separate the DNA samples with a size of several hundred to 20,000 base pairs (bp). DNA molecules are negatively charged so that in the electric field will migrate through the gel matrix toward the positive pole (anode). The larger the molecular size, the lower the rate of migration. The molecular weight of a DNA fragment can be estimated by comparing the rate of migration at the rate of migration of DNA fragments of molecular standards (DNA markers) of known size. Visulisasi DNA is then performed under ultraviolet light exposure after the first gel was added a solution of ethidium in the making bromid. Another way to see the visualization of DNA was gel immersed in a solution of ethidium bromid described above prior to ultraviolet rays.• Capillary ElectrophoresisElectrophoresis methods used to separate amino acids, proteins, lipids, carbohydrates, and nucleotides with a high resolution performed on capillary tube containing buffer. This method came into widespread use at the end of 1940 for applications in various fields such as biotechnology, chemical, environmental and pharmaceutical analysis. Capillary electrophoresis using a high-voltage electricity that causes all the components of ions or neutral molecules move to the cathode. Detection can be performed by spectrometry or electrochemical detection techniques. This separation technique is influenced by the electric voltage, the diffusion coefficient, length and diameter of the capillary tube, and the concentration of the sample. This method has good efficiency and selectivity, but wasteful of electricity because it uses high voltage and tools are also expensive.

 Schematic drawings of capillary electrophoresis
• Gel Electrophoresis KanjiFurther electrophoresis technique was developed to separate the larger biomolecules. 1955Smithies years demonstrate that the gel made of starch solution can be used to separate human serum proteins. The trick is to pour the hot starch solution into a plastic mold, after the starch is allowed to cool to form a solid gel but fragile. Starch gel acts as a stationary phase (stationary phase) on Whatman filter paper in the previous technique. It turns out that was introduced gel electrophoresis Smithies triggered scientists to find other chemicals that can be used as a gel material better, such as agarose and acrylamide polymers. Starch gel electrophoresis and discovery in the early career took him Smithies received the Nobel Prize in medicine in 2007.
Capillary Electrophoresis Process
The tools used in capillary electrophoresis is• capillary column (with silica, the optical window in order to observe the process dariluar)• Two electrodes.• Power supply (can be set for high-voltage)• Detector (UV rays)• buffer solution along with two storage places.
2.1.3 Function ElectrophoresisTo determine:• The molecular weight of a material• The number of types of proteins in a sample• The existence of fraud or material damage / contamination of materials• The presence of antibodies against specific viral or bacterial pathogen• protein isoelectric point• To produce industrial goods made of rubber. For example in the manufacture of dolls and gloves, rubber mold is deposited in the form of a glove puppet or electrophoresis.• To reduce air pollutants released from smokestacks. This method was developed by Frederick Cottrell (1877-1948) of the United States. Dalamdilengkapi the factory chimney with the "precipitating electrostatics" in the form of metal plates which are given an electrical charge, which will attract and agglomerate of fine dust in the exhaust fumes.
2.1.4 Principles of ElectrophoresisThe working principle of electrophoresis is based on the movement of negatively charged particles (anions), in the case of the DNA, which is moving toward the positive pole (anode), while the positively charged particles (cations) will move toward the negative pole (anode) (Klug & Cummings 1994: A-6). Electrophoresis was used to observe the amplification of DNA. Electrophoresis results are shown formed a band that is the amplification of DNA fragments and showed the pieces of the number of pairs basanya (Klug & Cummings 1994: 397).Electrophoresis technique that made use of the gel medium. Displacement of particles in the gel medium is influenced by factors such as particle size, composition and gel concentration, charge density, electric field strength and so on. The smaller the particle proficiency level, the movement or migration will be faster, because the gel matrix containing a complex network of pores so that the particles can move through the matrix (Brown 1992: 19).
 Working drawings Electrophoresis ProcedureThe working procedures in performing DNA electrophoresis using agarose gel electrophoresis technique as follows:Materials and Equipment:A. DNA markers, such as λ DNA cut with HindIII2. DNA samples, for example:3. Bacterial chromosomal DNA,4. Plasmid DNA isolation results (uncut)5. Plasmid DNA restriction results (cut)6. Agarose7. 50X TAE buffer solution (242 g of tris-base; 57.1 g of glacial acetic acid: 100 ml of 0.5 M EDTA pH 8; dissolved in distilled water to 1000 ml)8. Distilled water9. Measure 1000 ml glass10. 50 ml Erlenmeyer flask11. Tube mikrosentrifuga12. Gloves13. Micropipette with the tip set (Bio-Rad and Axygen Scientific)14. Parafilm paper15. A set of tools electrophoresis16. 6x loading dye (0.25% bromophenol blue; cyalol 0.25% xylene, 15% ficoll type 4000; 120 mM EDTA)17. Solution of ethidium Bromid (EtBr)18. UV transluminator19. UV glasses20. Digital cameras

How it WorksA. Make 250 ml of 1x TAE buffer by mencamnpurkan 5 ml 50x TAE in 245 ml distilled water into.2. Create a 1% agarose gel by weighing 0.2 g agarose to dissolve into a 1x TAE buffer until the volume of 20 ml. Agarose solution was boiled to dissolve completely.3. Prepare agarose gel tray, attach the tape at each end of an agarose gel tray (make sure that the tape attached to a strong and there is no hole at each end of the tray)4. Pairs of comb at one end of the electrophoresis agarose gel tray with a position almost touching the base tray5. Check the temperature agarose solution by attaching erlenmeyer to hand, if the temperature is down to around 50-60 0C, add 1 ml ethidium bromid (WARNING HARD!, Wear gloves because it is carcinogenic).6. Agarose solution was homogenized briefly, then pour the solution into the tray agarose gel, leave until the solution becomes a solid gel.7. Take the comb carefully, remove the tape from the ends of the tray.8. Insert the tray which already contains the agarose gel electrophoresis in a tank filled with a solution of 1x TAE buffer (make sure that the gel is completely submerged in TAE).9. Prepare about 5 cm near the parafilm paper electrophoresis tank.10. Enter a 10 ml sample of DNA and 2 ml 6x loading dye into agarose gel wells by mixing the two materials in advance evenly on the paper using a micropipette parafilm.11. Make a note of the number and type of DNA sample wells are included.12. Connect the cable from the current source to the electrophoresis tank (make sure that the cables connected to the negative pole is near the wells, if not, change the position of the tray / gel in the opposite direction).13. Turn on current source, set volatase and running time to obtain the 70 V and 45 minutes by pressing the appropriate button on the current source.14. Run electrophoresis (do running) by pressing the run button on the current source.15. Electrophoresis stops when set time is up, which is characterized by the presence of the alarm. Turn off the current source and remove the tray from the electrophoresis tank.16. Remove gel and place it on top of UV transluminator (put the glass envelope of black on the UV transluminator).17. Turn on the UV transluminator, observe the DNA bands that tervisualisasi