Like most other community analysis methods, TRFLP is also based on PCR amplification of a target gene. In the case of TRFLP, the amplification is performed with one or both the primers having their 5’ end labeled with a fluorescent molecule. In case both primers are labeled, different fluorescent dyes are required. While several common fluorescent dyes can be used for the purpose of tagging such as 6-carboxyfluorescein (6-FAM), ROX, carboxytetramethylrhodamine (TAMRA, a rhodamine-based dye), and [https://en.wiktionary.org/wiki/hexachlorofluorescein hexachlorofluorescein] (HEX), the most widely used dye is 6-FAM. The mixture of amplicons is then subjected to a restriction reaction, normally using a four-cutter restriction enzyme. Following the restriction reaction, the mixture of fragments is separated using either capillary or polyacrylamide electrophoresis in a DNA sequencer and the sizes of the different terminal fragments are determined by the fluorescence detector. Because the excised mixture of amplicons is analyzed in a sequencer, only the terminal fragments (i.e. the labeled end or ends of the amplicon) are read while all other fragments are ignored. Thus, T-RFLP is different from ARDRA and RFLP in which all restriction fragments are visualized. In addition to these steps the TRFLP protocol often includes a cleanup of the PCR products prior to the restriction and in case a capillary electrophoresis is used a desalting stage is also performed prior to running the sample.