BME100 f2018:Group7 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 Error creating thumbnail: File missing Name: Daniella McFeely Role: Innovative Researcher Error creating thumbnail: File missing Name: Jennifer Smetanick Role: Innovative Researcher Error creating thumbnail: File missing Name: Ethan Armendariz Role: Innovative Researcher Error creating thumbnail: File missing Name: Sebastian Tellez Role: Innovative Researcher LAB 5 WRITE-UP PCR Reaction Report Since we were rather new at micropipetting, all team members contributed to micropipetting the samples so we could all gain that experience. We were all able to pipette without too much issue. The pre-lab reading and instructional video proved instrumental for us to complete this part of the lab. With this prior knowledge, we were able to pipette correctly and verify that all group members were pipetting correctly as well. Before we used the pipette, we each tested the micropipettor to identify the first and second stop. This way we would be confident when actually pipetting the solution. The final reactions had a similar amount of liquid, but some tubes had slightly more liquid than other. We attributed this to minor error in pipette technique such as: releasing from the pipette stop too quickly and getting air bubbles. There was no liquid left in the tubes, but there is possibility that small amounts, not visible to the eye, remained. We did not have to change our initial labeling scheme. The pipetting section went smoothly and the tubes were then ready to be placed in the OpenPCR machine. Once we had another group to share the machine, the settings were marked and the machine was closed and started. After the cycles were complete, the samples were preserved in the freezer until our next lab. Fluorimeter Procedure Imaging set-up 1. Identify a smartphone camera to take pictures with (we used a Samsung phone) and set the recommended camera settings: Inactivate the flash Set ISO to 800 or higher Set white balance to auto Set exposure to highest setting Set saturation to highest setting Set contrast to lowest setting 2. Place the phone in the cradle 3. Adjust height of fluorimeter so that the camera aligns with the slide. We added three trays to the bottom of the fluorimeter and placed the smartphone 5.0 cm away from the fluorimeter so the images would be close enough to the sample and keep the image in focus. 4. Set the camera timer to three seconds to allow ample time to close the lightbox lid for the picture. Placing Samples onto the Fluorimeter 1. Insert a slide in the fluorimeter with the smooth glass side down 2. Turn on the excitation/blue LED light. 3. Use a micropipette to drop 80 microliters of SYBR Green I in between the first two dots of the slide and is centered with the excitation light. 4. Micropipette an 80 microliter drop of the sample/calibration solution on top of the drop of SYBR Green I. 5. Start camera timer and lower lid of the lightbox in time for the picture. 6. Repeat step 6 for a total of three pictures. 7. Open lid and micropipette sample and dispose in the designated waste container 8. Move the slide to the next position for a new sample. 9. Repeat from step 3-8 for all other samples. Next Step Use ImageJ to analyze these images and draw a final conclusion for each patient. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA High: 5 μg/mL sample' Error creating thumbnail: File missing Low: 0.5 μg/mL sample Error creating thumbnail: File missing 0 μg/mL sample Error creating thumbnail: File missing Calibrator Mean Values Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND ' ' MEAN Standard Deviation Image 1 Image 2 Image 3 5 2.5 C-1 4542918 4558634 4520038 4540530 19408.49587 2 1 C-2 5384193 5438443 5424641 5415759 28194.55742 1 0.5 C-3 3646884 3564298 3614559 3608580.333 41616.34523 0.5 0.25 C-4 4824435 4838666 4814922 4826007.667 11949.86796 0.25 0.125 C-5 3887120 4014102 4164017 4021746.333 138606.6882 0 0 C-6 1300777 1293316 1305037 1299710 5932.902072 Calibration curves Error creating thumbnail: File missing includes all data points Error creating thumbnail: File missing without highest data point value Images of Our PCR Negative and Positive Controls Negative Control Error creating thumbnail: File missing Positive Control Error creating thumbnail: File missing PCR Results: PCR concentrations solved PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration (µg /mL) G7 + 5262558.667 2.262558667 12 27.150704 G7 - 846462 -2.153538 12 -25.842456 G7 1-1 5478185.667 2.478185667 12 29.738228 G7 1-2 3847307.667 0.847307667 12 10.167692 G7 1-3 5144280.667 2.144280667 12 25.731368 G7 2-1 2077352.333 -0.922647667 12 -11.071772 G7 2-2 1422916.667 -1.577083333 12 -18.925 G7 2-3 1623531.333 -1.376468667 12 -16.517624 PCR Results: Summary Our positive control PCR result was 27.15 μg/mL. Our negative control PCR result was -25.84 μg/mL. Observed results Patient 28063 : The image has a greener tint, hinting that there is a statistically significant amount of DNA present in the solution. The three samples for patient 28063 reflects the qualitative conclusion and is most similar to the positive control. Each trial has the following quantities in μg/mL: (1) 29.74, (2) 10.16, and (3) 25.73. Patient 28963 Sample 1 Error creating thumbnail: File missing Patient 67655 : The image lacks much of a green tint, hinting that there is not a statistically significant amount of DNA present in the solution. The three samples for patient 67655 reflects the qualitative conclusion and is most similar to the negative control Each trial has the following quantities in μg/mL: (1) -11.07, (2) -18.93, and (3) -16.52. Patient 67655 Sample 2 Error creating thumbnail: File missing Conclusions Patient 28063 : Each sample for patient 28063 is most similar to the positive control of 27.15 μg/mL. The average of the three samples is 21.88 μg/mL. Because this value is very similar to the positive control, one can conclude that patient 28063 is the positive control. Patient 67655 : Each sample for patient two is most similar to the negative control of -25.84 μg/mL. The average of the three samples is -15.51 μg/mL. Because this value is most similar to the negative control, one can conclude that patient 67655 is the negative control.