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NAU BIO 205L - BIO205_Prelab8

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Kristina SimonovicSection O9 March 2015Exercise EightTitle:Bacterial Molecular Identification (PCR)Hypothesis:If a PCR test is properly conducted, then the unknown’s identity will be confirmed through analysis of extracted DNA fragments.Methods:Transfer the specified volumes from each reagent tube located in the floaty into one of the four 200 microliter tubes in the strip. After all tubes are prepared, the strip should be spun down in themicrocentrifuge. From the 24-hour culture plate, a single isolated colony is selected and, using the end of a 10 microliter pipette tip, lightly touched leaving a very slight dimple on the outside edge of the selected colony. The culture on the 10 microliter pipette tip containing the E.I. cells should be transferred into a 200 microliter microcentrifuge tube containing the combined PCR reagents. DO NOT spin the tube down again. Now the strip of 200 microliter tubes can be placedinto the thermal cycler. This thermal cycler program will be run for approximately 1 hour. Duringthis time, you will finish the exercise lecture and prepare a 1% agarose gel for electrophoresis of the reactions to determine if there is product. The gel you pour will have 6 wells in it that were created by the gel comb. To check that the PCR reaction was successful and to determine the sizeof the DNA product fragment, load 7 microliters of the product from each of the PCR reactions loaded into the 1st, 2nd, 3rd, or 4th lanes of the 1% agarose gel and then run at 300 volts for 12 minutes. If PCR product is present, a band of DNA of the appropriate size will be seen in the finished gel when placed on the transilluminator. It is important to record in which tubes product is detected on the reaction tube plate sheet provided in each lab. The remaining 16S rDNA PCR product will be used for a cycle sequencing reaction and sequenced using a 3730XL Sanger sequencer at our in house sequencing facility EnGGen. When the sequencing is complete your E.I.s 16S sequence will be sent to you along with the instructions of how to determine its quality,trim the sequence and submit it to BLAST on the NCBI website to determine the unknowns identity. Safety Concerns:1. This exercise requires keen attention to detail and technique for the accurate transfer of the reagents employed in a PCR reaction.2. Fresh culture is very important in obtaining product from the PCR reaction3. Gloves should be worn throughout this experiment4. Safety goggles and lab coat are necessary at all timesRelevance:Conducting a PCR test will determine the unknown isolate’s identity by analyzing the extracted DNA

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