DNA Extraction and Polymerase Chain Reaction
DNA Extraction:
DNA extraction was accomplished by following the protocol given in the ZR Fecal DNA Mini-Prep kit from Zymo Research. All the buffers, solutions, tubes, and columns necessary for DNA extraction were provided in the kit. The protocol was followed. To find the concentration of DNA in the resulting solution, the sample was run through a Thermo Scientific Nanodrop 2000 Spectrophotometer.
Polymerase Chain Reaction (PCR):
A successful PCR test requires Taq polymerase, different primers, nuclease free water, and the extracted DNA Six PCR tubes were labeled 1-6. In each PCR tube, 12.5µL of the Taq Polymerase was added. Then, in tubes 1 and 2, the 515F/806R primer was added. 2µL of the commonly used 18S fungal rDNA primer, NS1/NS2, was added to tubes 3 and 4 while 2µL of the 533F/1492R primer was added to tubes 5 and 6. Tubes 1, 3, and 5 din not contain DNA to serve as a controls for each primer. Therefore, the only tubes that contain DNA are tubes 2, 4, and 6. In tube 2, 6.1µL of DNA was added, 5.0µL of DNA was added to tube 4, and 5.7µL of DNA was added to tube 6. All reactions were brought to a final volume of 25uL using Nuclease free water. Once all of the DNA was added, Each tube was placed into a Thermo-cycler. Once the DNA is placed into the Thermo-Cycler, the Thermo-Cycler increased the temperature to 94ᵒC for one minute for the initial denaturing of the DNA. The Thermo-Cycler kept the temperature at 94ᵒC for another 15 seconds to denature the DNA. After 15 seconds, the temperature decreased to 55ᵒC for 15 seconds to allow for the primers to anneal to the denature DNA. The temperature was then raised to 72ᵒC to all for the DNA polymerase to extend and replicate the DNA by adding complementary nucleotides to the template strand for 30 seconds . Then the Thermo-Cycler goes back up to 94ᵒC and repeats the entire cycle 35 times. After 35 cycles, the temperature was raised to 72 degrees one more time for seven minutes for the final extension. For the second PCR reaction, the annealing temperature was raised to 60ᵒC instead of 55ᵒC.