Purpose: To be familiarized with the science and techniques used to identify different types of bacteria based on their DNA sequences.
Background Information: The process begins with preparing a sample. Successful identification starts with using a sample that is considered to be good. The first step is to pick up a single colony and drop it into a microcentrifuge tube. A buffer is used to dissolve the cell wall in order to extract bacterial DNA. This step may take several hours. The proteolytic enzymes need to go before proceeding. Heating the sample in a water bath at 100 degrees Celsius denatures them. Next, cellular debris is spun down in the centrifuge and appears as a pellet at the bottom. The DNA is contained in the liquid, which is then transferred to the tube. To continue the process, PCR amplification is conducted. One must add PCR Master Mix solution to the sample DNA to prepare the polymerase chain reaction. The mix contains water, a buffer to keep the correct pH for the reaction; large quantities of the four nucleotides; large quantities of oligonucleotide DNA primers; and a heat-stable DNA polymerase. At the same time, one will prepare negative and positive control reactions. The positive contains positive control DNA while the negative contains sterile deionized water. Both contain the PCR solution. Once reaction tubes have been loaded onto the PCR machine, DNA replication starts. By doing this, one can know temperature, time remaining, cycle number, melt, anneal, and extend. The first step, melt, is to separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95 degrees Celsius for 30 seconds. The vial is cooled at 60 degrees Celsius. The final step, extend, is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70 degrees Celsius for 45 seconds. Separation of the strands, annealing the primer to the template, and the synthesis of new strands