BME100 f2016:Group5 W1030AM L5

BME 100 Fall 2016	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: Tanner Henson Biomedical Engineering freshman, SNP finder and primer designer Name: Kevin Thieu Biomedical Engineering freshman, Background Research SNP and Assisted in OpenPCR Program Procedure Name: Shadi Farmawi Biomedical Engineering freshman, Researcher Name: Cindy Wong Biomedical Engineering Freshman, Researcher Name: student Role(s) LAB 5 WRITE-UP PCR Reaction Report Report The pipetting for this lab was very easy overall, after a few cycles of the pipetting, it became very easy and it was almost like muscle memory.The pre-lab reading aided greatly in understanding the use of lab equipment specifically in the use of the micropipets. The difference between the first stop and the second stop on the micropipetting is the first stop is used to suck up the liquid and the second stop of the pipette is used to shoot the liquid out of the tip of the pipette.The final reactions of the PCR had the same amount of liquid, however the concentration of the DNA within the liquid increased, the actual volume of the liquid did not increase.The tubes appeared to be clean and dry with no visible signs of contamination. We did not have to change our labeling scheme since there were no complications that would have required such an action. Fluorimeter Procedure Imaging set-up To set up our device, a Samsung Galaxy s7, to capture images from the fluorimeter, we had to first set up the camera. The camera's settings were changed to have a film speed, or ISO of 800. Also, the camera flash was inactivated, white balance was changed to auto, exposure and saturation were at its highest setting, and contrast was at its lowest setting. placed the phone on a given smart phone cradle. The smart phone cradle should be at a right angle and positioned where the camera would take images sideways. Next, set the cradle exactly four centimeters away from the drop. From there, pictures can be taken from the fluorimeter. Placing Samples onto the Fluorimeter Set the micropipette to 160 microliters and drop 160 microliters of water on the slide. Make sure to position it in the middle first two rows to ensure that the drop looks like a beach ball. Use a smartphone that has been calibrated to its necessary settings, take three consecutive pictures of the drop. Dispose of the tip and tray Set the micropipette to 80 microliters and drop 80 microliters of SYBR Green I in the middle first two rows to ensure that the drop looks like a beach ball. Add 80 microliters of calf thymus (or water blank) Adjust the blue LED light to focus on the drop in the middle Use a smartphone that has been calibrated to its necessary settings, take three consecutive pictures of the drop Remove the 160 microliter of sample from the surface using a micropipettor Repeat steps 4-8 for the other concentrations of Calf Thymas DNA Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Zero DNA .5 μg/mL DNA 5 μg/mL DNA Calibrator Mean Values Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL) Final DNA Concentration in SYBR Green 1 Solution (ug/mL) Sample Number RAWINTDEN DROP- BACKGROUND Image 1 Image 2 Image 3 MEAN Standard Deviation 5 2.5 C-1 25532876 28695597 28026721 27418398 1666806.433 2 .1 C-2 12026091 12603902 12665434 12431809 352706.4904 1 .5 C-3 18619158 18554353 19162023 18778511.33 333707.6877 .5 .25 C-4 21133802 22926384 22447325 22169167 928093.4751 .25 .125 C-5 19449990 19305835 18617074 19124299.67 445144.4414 0 0 C-6 5673222 5295378 5155843 5374814.333 267680.5105 Calibration curves Name: Dot Plot 1 (w outlier point) Name: Dot Plot 2 (w/o outlier point) Images of Our PCR Negative and Positive Controls Negative Control 1 Negative Control 2 Negative Control 3 Positive Control 1 Positive Control 2 Positive Control 3 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) PC 10751434.33 -1.313869938 12 -15.76643926 NC 9771507 -1.713477404 12 -20.56172885 1-1 7885116.333 -2.482734237 12 -29.79281084 1-2 8877497.333 -2.078048252 12 -24.93657902 1-3 8614098 -2.185460645 12 -26.22552774 2-1 8421506.667 -2.263998035 12 -27.16797642 2-2 10837873.67 -1.278620584 12 -15.34344701 2-3 9702029.333 -1.741809907 12 -20.90171889 PCR Results: Summary Our positive control PCR result was -15.7664 μg/mL Our negative control PCR result was -20.5617 μg/mL Observed results Patient 49665: Qualitative: Bubbly beach ball like structure Quantitative: Trial 1 = -29.79281084(μg/mL), Trial 2 = -24.93657902(μg/mL), Trial 3 = -26.22552774 Patient 36290: Qualitative: Bubbly beach ball like structure Quantitative: Trial 1 = -27.16797642(μg/mL), Trial 2 = -15.34344701(μg/mL), Trial 3 = -20.90171889 Conclusions Patient 49665: Negative Patient sample demonstrates a concentration reading that is closer to the negative control than the positive control while remaining consistent. Patient 36290: Inconclusive Patient sample demonstrates a concentration reading that is inconsistent with data varying greatly depending on different trials yielding inconclusive results.