BME100 f2018:Group3 T1030 L5

BME 100 Fall 2018	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Taylin Aasen Name: Jaqueline Bello Mendoza Name: Joan Ponce Sanchez Name: Kaprao Fuegner LAB 5 WRITE-UP PCR Reaction Report Required 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. Tube Label PCR Reaction Sample Patient ID G3 + Positive control none G3 - Negative control none G3 1-1 Patient 1, replicate 1 49652 G3 1-2 Patient 1, replicate 2 49652 G3 1-3 Patient 1, replicate 3 49652 G3 2-1 Patient 2, replicate 1 99085 G3 2-2 Patient 2, replicate 2 99085 G3 2-3 Patient 2, replicate 3 99085 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 Procedure Imaging set-up To set up the device, place the flourimeter onto the table. Then identify the rough side of the slide, place the slide onto the flourimeter with the rough side facing upwards. Turn on the flourimeter and adjust the slide so that the light passes through two rows of circles. Set the camera about 4 cm away from the light/drop. Inactivate the flash and set a countdown timer to 3 seconds. Placing Samples onto the Fluorimeter 1. Placed a clean strip to the fluorimeter 2. Place the pipette with correct measurement 3. Organized the tubs so we wouldn't get confused 4. Began pipetting a sample at a time and used half of the strip for one sample and the other have with another 5. Every time the drop was aimed to have filled two circle on the strip 6. Took 3 photos per sample and noted down the corresponding samples to photos 7. Replace the tip of the pipette each time 8. Every time the sample drop was aimed to have filled two circle on the strip 9. Covered the flurimeter with a lid to capture the result Materials: - 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 Analysis Images of High, Low, and Zero Calf Thymus DNA High 5-1 Sample Low 0.5 -1 Sample Zero H2O sample Calibrator Mean Values Calibration curves Images of Our PCR Negative and Positive Controls Positive Control Negative Control PCR Results: PCR concentrations solved PCR Results: Summary Our positive control PCR result was 64.77 μg/mL Our negative control PCR result was 13.55 μg/mL Observed results Patient 49652 : When splitting the colors apart using the image J the contrast was a huge help to identify the bigger dark figure inside the drop. The result of this patient was the given concentration of 60.64μg/mL, 17.11μg/mL, and 18.22μg/mL. Patient 99085 :When splitting the colors apart using the image J there is allot of contrast which helped to see the inner small shape figure. With color it was a bright green with darker green inside. The concentration of the PCR of this patient resulted in 16.06μg/mL, 0.46μg/mL,-7.48μg/mL. Conclusions Patient Negative, due to two out of the three values being extremely similar to the given negative values. The single positive value being out weighted by what the other evidence supports. Patient Negative, due to the fact that all three results stay more inline to the given negative value when compared.