Unknown Mixed Microorganisms

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Unknown mixed microorganisms were collected from a building sample, air and fingerprints. The sample microorganisms were swabbed onto petri dishes that contained growth mediums: Vancomycin, EMB- lactose and PEA. They were placed in an incubator at 37º C for 1-2 days to allow growth on the mediums. The incubated microorganisms were observed to determine whether the samples were bacteria or fungi. The samples that had the bacterial colony morphology were selected for the study. Liquid cultures of the selected bacterial colony were prepared for the PCR amplification of the 16S ribosomal RNA. Two PCR tubes: tube B and tube C, were prepared with each containing 2X PCR Master Mix. Into each tube, 20 µl of 16S RNA primer mix were added. Tube B was selected to contain the prepared bacterial DNA and 5 µl of the liquid DNA was added. Tube C was the selected control tube. It didn’t contain any bacterial DNA and to make up for the absence of the DNA µl, 5µl of sterile water were added to this tube. Both tubes were brought to a total volume of 50 µl. The mixture was kept…show more content…
They were run at 1X 94ºC for 3 minutes, 30X at 94ºC for 30 seconds; 50ºC for 30 seconds; 72ºC for 45 second and 1X at 72ºC for 5 minutes. The PCR reactions took about 1 hour and 30 minutes to complete. The PCR products, were then purified by removing the leftover primers, nucleotides and salts. 250 µl of Buffer BB were added to Tube B and the mixture was pipetted into a spin column. The mixture was centrifuged for 30 seconds at room temperature. Then 2 cycles were completed at 30 seconds each with 200 µl of Buffer WB to remove any impurities. Then 25µl of Buffer EB were added to the tube to release the pure DNA and the mixture was centrifuged for 30 seconds. As the PCR reaction was running, a microscope slide was prepared from the live bacterial culture to observe the individual cells of the unknown bacteria and determine its
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