BME100 f2018:Group3 T1030 L5
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OUR TEAMLAB 5 WRITE-UPPCR Reaction ReportRequired Materials: -Empty PCR Tubes -Scissors -Sharpie -Cup for used pipette tip -Micropipette -Disposable pipette tip -Tube holding container --------------- -PCR reaction mix -Patient + positive and negative control DNA primer During the lab we transferred 50 micro-liters of PCR reaction mix into the PCR tube using the micropipette with a disposable tip. Dispose the tip into the cup. Using a new pipette tip, we transfered 50 microliters of the positive DNA Primer into the same tube. Dispose of the tip and label the tube accordingly. We did this for the negative and patient DNA Primer. When all the tubes are filled with DNA primer and PCR reaction mix and properly labeled, the PCR Tubes are then transferred to the thermocycler to for the heating and cooling process. All groups members discarded the used pipette tips and returned all supplies.
Thermocycler Steps: 1.INITIAL STEP: 95°C for 2 minutes: 2.Denature at 95°C for 30 seconds: 3.Anneal at 57°C for 30 seconds: 4.Extend at 72°C for seconds: 5.FINAL STEP: 72°C for 2 minutes: 6.FINAL HOLD: 4°C Fluorimeter ProcedureImaging set-up
Placing Samples onto the Fluorimeter
- 2 tubes of 1000 μL of SYBR Green 1 Solution. - 8 tubes of 500 μL of Buffer. - 1 tube of 1000 μL of water at pH=8 - 5 tubes labeled 0.25, 0.5, 1, 2, and 5. These contain Calf Thymus DNA Procedure: 1. Turn on the blue LED light and position the slide so that the light shines in between 2 rows of circle 2. Unwrap the SYBR Green I Solution from the aluminum foil. 3. Using the pipette, place down 80 μL of the solution in between the rows of circles where the light passes through. Then quickly rewrap the solution. 4. Using a new pipette tip, add 80 μL of the Calf Thymus Solution, labeled 5, to the SYBR Green I solution on the slide. 5. Cover the setup with a box and focus the camera onto the droplet. 6. Close the box to ensure no light penetrates and capture at least 3 images of the droplet. 7. Remove the box and dispose of the solution and slide. 8. Repeat steps 1-7 for other Calf Thymus Solution and the PCR Solution from previous lab
Data Collection and AnalysisImages of High, Low, and Zero Calf Thymus DNA High 5-1 Sample Low 0.5 -1 Sample Zero H2O sample
Calibration curves
PCR Results: PCR concentrations solved
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