BME100 f2018:Group14 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: Carlos Matherson Role: Resident Lab Dude Name: Michael Esposito Role: SNP Researcher Name: Mohamed Sorkati Role(s) Name: Branden Dora Role(s) [1] Name: Daniel Bhella Role(s) LAB 5 WRITE-UP PCR Reaction Report The pre-lab reading definitely improved our micro-pipetting technique, and aided in our overall understanding of what we were accomplishing by moving substances to and from test tubes. Being able to understand the difference between the first and second stop on the pipettor helped us prepare more accurate solutions, which would make for more ideal and accurate results. The experienced technique we developed by thoroughly reading the pre-lab material allowed us to pipette the liquids so that the test tubes all had nearly, if not exactly, the same amount of liquid in them. Being precise in the amount of specific liquids in the test tubes was important because of the interactions that needed to take place between the fluids. For example, if the ratio of SYBR Green dye to Calf Thymus DNA is skewed, then the results obtained in ImageJ would not be accurate. Another area in which precision was important was the labeling of the test tubes. If one patient's DNA sample was mislabeled and confused with the positive control, then this patient would have been misdiagnosed as having the diseased SNP and the results would have been invalid. Fluorimeter Procedure Imaging set-up To set up the imaging for the DNA samples, we obtained a light-obstructing box, a fluorimeter, two closed and empty test tube racks (to stand the fluorimeter on), a sufficient amount of slides, a smartphone camera, and a stand to place the smartphone in. The smartphone camera was set to have a five second timer, the highest ISO setting at 800, the highest exposure setting at 1/4 s, the highest saturation setting, the lowest contrast setting, and the flash turned off. Had the camera we used not had changeable settings, the default settings would have sufficed, as long as the flash was disabled, and a 3-10 second timer was enabled. Additionally, three pictures of each sample must be taken. Another important part in setting up the imaging was obtaining the correct camera angle. For the most effective measuring of the fluorescence, the camera must be angled so that the image captured is a perfect side view of the drop of liquid. In other words, the lens of the camera should be perpendicular to the textured face of the slide. Placing Samples onto the Fluorimeter Place a new tip on the micropipettor. Obtain 80 μL of SYBR Green, and put it on the slide, in the center of the middle rows of viewing windows. Place a new tip on the micropipettor. Obtain 80 μL of one of the Calf Thymus DNA solutions, or the water. Put this liquid on top of the drop of SYBR Green. Align the slide so that the light from the fluorimeter is in the center of the drop. Move the fluorimeter into the light-obstructing box. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA High Concentration Calf Thymus DNA Low Concentration Calf Thymus DNA Zero DNA 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 5004907 4812356 4661229 4826164 172254.572 2 1 C-2 5018667 4898470 5102159 5006432 102394.2069 1 0.5 C-3 3891886 3714357 3540593 3715612 175649.8626 0.5 0.25 C-4 5029228 5028016 5037606 5031616.667 5222.195068 0.25 0.125 C-5 3405909 3408662 3411234 3408601.667 2663.012642 0 0 C-6 7587604 7570325 7723295 7627074.667 83775.92357 Calibration curves Images of Our PCR Negative and Positive Controls Negative Control Positive Control PCR Results: PCR concentrations solved PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) "PCR Product Concentration (µg /mL)(Step 5 calculation)" Total Dilution "Initial PCR Product Concentration (µg /mL) (Step 6 calculation)" G14 + 19181505 30.82827855 12 369.9393426 G14 - 1786266 -4.425647563 12 -53.10777075 G14 1-1 2029143.67 -3.933421418 12 -47.20105702 G14 1-2 1946893.67 -4.100112742 12 -49.20135291 G14 1-3 2278082.67 -3.428911126 12 -41.14693351 G14 2-1 3683874.67 -0.5798736794 12 -6.958484153 G14 2-2 2642036.67 -2.691306576 12 -32.29567892 G14 2-3 1583786 -4.836002083 12 -58.032025 PCR Results: Summary Our positive control PCR result was 369.94 μg/mL Our negative control PCR result was -53.11 μg/mL Observed results Patient 79929 : The solution was not fluorescent. The average concentration for this patient was -49.84 μg/mL, which is closer to the negative control. Patient 84911 : The solution was not fluorescent. Therefore, the patient was concluded to be negative. The average concentration for this patient was -26.8μg/mL, which is closer to the negative control. Conclusions Patient 79929 : The results determined that this patient is negative because the concentration in the patient's sample was closer to the negative control than the positive control. Patient 84911 : The results determined that this patient is negative because the concentration in the patient's sample was closer to the negative control than the positive control.