more effective. This system is specifically designed to separate DNA fragments larger than 1Mb, making it a powerful tool for larger DNA analysis. Protocol The protocol typically involves the following steps: Preparing the agarose gel: An agarose gel is prepared by mixing agarose powder with TBE buffer and heating the mixture to dissolve the agarose. Agarose is a polysaccharide that forms a gel when it is dissolved in a buffer and solidified. TBE buffer (Tris-Borate-EDTA) is commonly used in PFGE as it helps to maintain the integrity of the DNA during electrophoresis. The gel is poured into a casting tray and allowed to solidify. Once solidified, the gel is inserted into the electrophoresis chamber. Loading the DNA sample: The DNA sample is digested with a restriction enzyme to generate a set of fragments of known size. Restriction enzymes are enzymes that cut DNA at specific sequences. The fragments are then loaded onto the gel, typically in a well that is created by punching a hole in the gel. This well is used to load the sample and the sample will be separated by different fragment size in the gel Running the electrophoresis: The gel is placed in the electrophoresis chamber, which is filled with TBE buffer. A pulsed electric field is applied to the gel, which causes the DNA fragments to migrate through the gel. The time of the electrophoresis is generally between 18 to 48 hours. The principle of this electrophoresis is that DNA fragments will move in the gel under an electric field and migrate towards the anode. Larger DNA fragments will move more slowly than smaller ones, allowing them to be separated by size Staining the gel: Once the electrophoresis is complete, the gel is removed from the chamber and stained with a DNA-specific stain, such as ethidium bromide. Ethidium bromide is a fluorescent dye that intercalates into DNA and causes the DNA to fluoresce when exposed to UV light. The stained gel is then visualized under UV light to identify the DNA bands. The final gel will show different bands of different size, the bigger band is representing larger fragment size. Applications PFGE may be used for genotyping or genetic fingerprinting. It has commonly been considered a gold standard in epidemiological studies of pathogenic organisms for several decades. For instance, subtyping bacterial isolates with this method has made it easier to discriminate among strains of Listeria