Yeager

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School

Michigan State University *

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141

Subject

Biology

Date

Dec 6, 2023

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pdf

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Uploaded by BrigadierBarracuda3621

BS171 1 Research Experiment 2: Non-cultural Detection of Bacterial Traits Prelab Name: Makayla Yeager Date:10/31/23 Purpose Determine if the Potter Park Zoo increases the presence of tetracycline - resistant bacteria and other pathogenic bacteria in the river. Experimental Design Figure 1: Non-Cultural Identification of Bacterial Characteristics. This outlines the non-cultural approach for identifying bacterial traits. It encompasses sequential stages such as filtration to isolate bacterial cells, followed by DNA extraction. As well as a master cocktail solution is prepared, which contains the necessary reagents for PCR. This master cocktail is then subjected to thermal cycling in a specialized device known as a thermocycler. Finally, the amplified DNA fragments are separated by size using agarose gel electrophoresis to observe the band on the gel. Manipulated variable: water sample site (upstream/downstream) Response variable: The band of expected size on the gel Negative Control: purified H2O, a sample that does not contain the gene you are amplifying. Positive Control: a sample that contains the tetB you are amplifying. Protocols Harvest of whole river communities by filtration Our assigned sample for harvest was Downstream B Harvesting river microbial communities by filtration 1. Record the sample your team is processing here Downstream B 2. Shake the sample well to re-suspend any sediment or particulate matter.

BS171 2 3. Measure out 1 L of your water sample using a graduated cylinder. 4. Transfer that 1 L of your sample to the filter unit. 5. Connect the filter unit to the vacuum hose and turn on the vacuum until all of the liquid has passed through the filter. 5. Turn off the vacuum and disconnect the hose. 6. Unscrew the collection bottle and pour the filtrate down the sink. 7. Screw the filter back together, measure out a second liter of water and filter it. a. Have your instructor evaluate the flow rate of your second liter to determine if you should filter additional water. 8. Record the total volume filtered here (fill in your ELN): Final weight. 6.7077 g - Tare weight 6.5903 g ______________________ Final weight 0.1174 g Extracting DNA from your filtered samples Community DNA was extracted from water samples collected upstream and downstream of the Potter Park Zoo using the Qiagen DNA Purification Kit ™ per the manufacturer’s protocols. Measuring DNA yields using a UV spectrophotometer 1. Remove the cover from the BioDrop instrument and press the power button to turn it on. 2. Select the Life Sciences icon --> Nucleic acid symbol icon --> Select DNA icon --> Select the DNA boxes and click the forward arrow on two screens to see the screen with all the data. 3. Add 10 μL of nuclease-free water to the sample depression to rinse, then remove with a kimwipe. 4. Blank the spectrophotometer: a. Add 1.5 μL of C6 solution to the sample depression. b. Click the blue cuvette icon to blank (zero) the machine. c. Use a kimwipe to remove the C6 solution. 5. Measure your first DNA sample: a. Add 1.5 μL of the first DNA sample to the sample depression. b. Click the red cuvette icon to read. c. Click the arrow, then click on the spectrum icon to go back to see the absorbance spectrum and concentration. d. Record the DNA concentration (mg/mL), the A260/A230 value, and the A260/A280 value. e. Click on the arrow and click on the spectrum again to go back to the data screen. f. Use a kimwipe to remove the sample. g. Rinse with 10 μL of nuclease-free water. 6. Repeat step 5 with the remaining DNA samples. 7. When done, press the X, do not save data (press X), then use the back arrow to get back to the main menu. 8. Press the power button to turn off and replace the cover Table 1. DNA concentrations and purity measurements. Sample [DNA] (ng/μL) A260/A230 A260/A280 Upstream DNA Downstream DNA

BS171 3 PCR amplification of selected genes Table 2. Single Reaction and Master Cocktail Volumes. Table 3. Single Reaction and Master Cocktail Volumes. Making the master cocktail 1. Calculate the volume needed for each ingredient and enter it into the last column of your Master Cocktail recipe table 2. Generate each master cocktail in a 0.5 mL tube using the green PCR reagent and make sure you have the right primer set for the gene that you want to test. Remember to always keep all reagents and your master cocktail on ice. 3. Using an ultrafine Sharpie, label your PCR tubes on the tops and upper side of the tube as shown in Figure 2 . Label the 4th tube with your Team ID and the primer pair being tested. 4. Vortex the master cocktail and aliquot 20 L of the master cocktail into each PCR tube. Check that the volumes in μ the tubes are identical. 5. Add 5 L of the appropriate DNA template at 5 ng/ L (25 ng total DNA) or μ purified water to the appropriate PCR tube. After adding to the tube, pipette up and down to ensure that the sample is added. 6. Make sure your PCR tubes are tightly and completely closed to avoid evaporation in the thermocycler. 7. Mix your PCR reactions by gently flicking the tubes in the strip. You may wish to briefly centrifuge the strip to ensure all the liquid is at the bottom of the tube. 8. Place your strip of PCR tubes in the thermocycler to run your PCR reactions. 9. Be sure to use the correct thermocycler program for the primer pair you are using. Reagent Single Reaction Volume Master Cocktail for 4 Reactions Volume Purified H 2 O 3.5 μL 14 μL PCR Reagent (buffer, dNTPs, Taq ) 12.5 μL 50 μL Forward Primer (@ 10 nM) 2 μL 8 μL Reverse Primer (@ 10 nM) 2 μL 8 μL Total volume 20 μ L 80 μ L Reagent Single Reaction Volume Master Cocktail for 5 Reactions Volume Purified H 2 O 3.5 μL 17.5 μL PCR Reagent (buffer, dNTPs, Taq ) 12.5 μL 62.5 μL Forward Primer (@ 10 nM) 2 μL 10 μL Reverse Primer (@ 10 nM) 2 μL 10 μL Total volume 20 μ L 100 μ L

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