BME100 f2016:Group15 W8AM 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: Adam Akkad Name: Shad Boswell Name: Nathan Hui Name: Carli Winchester LAB 5 WRITE-UP PCR Reaction Report The pipetting and PCR process was relatively smooth. We understood the that the difference between the first and second stop on the pipettor was that the first stop is used to absorb the liquid into the pipettor and the second stop is pressed to eject the liquid out. Our final reactions had the same amount of liquid as before the reaction. There was no liquid left in the DNA samples or PCR reaction mix. Our labeling scheme was consistent--it did not require any changes. Fluorimeter Procedure Imaging set-up First, we set up our smartphone camera vertically and placed it in the camera holder provided for us. We placed the slide onto the fluorimeter with the smooth side down. We then elevated the fluorimeter using several trays so that the camera was level with the slide on the fluorimeter. The camera was set to a timer of 5 seconds. After pipetting the fluorescent liquid and the solution we were measuring onto the slide, we took the picture and covered the phone and fluorimeter with a box so the picture could see the fluorescence more clearly. Placing Samples onto the Fluorimeter It should first be noted that all of the drops must be placed on a fluorimeter slide. In order to ensure that proper bubbles are formed, the rough side of the slide should be the side facing upwards. Beginning with the SYBR GREEN 1 solution, we added 80 uL to the rough side of the slide. Following this, 80 uL of H2O were added right on top of the SYBR GREEN 1 solution. This gave us a clear bubble which we could analyze. The blue LED light was then turned on. Adjustments were made to make sure that the light went directly through the middle of the solution bubble. The box was lowered to cover the fluorimeter to allow for proper lighting, after which 3 separate images were taken of the solution bubble. This process was repeated for all of the Calf Thymus DNA concentrations, as well as the PCR DNA mix from the previous lab. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA 1. 5 μg/mL sample 2. 0.5 μg/mL sample 3. 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 66323 66323 66323 66323 0 2 1 C-2 286393 286393 286393 286393 0 1 0.5 C-3 327772 327772 327772 327772 0 0.5 0.25 C-4 341592 341592 341592 341592 0 0.25 0.125 C-5 280596 280596 280596 280596 0 0 0 C-6 302079 302079 302079 302079 0 Calibration curves Images of Our PCR Negative and Positive Controls Positive Negative 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) Patient 1.1 435207 1.937217099 12 23.24660518 Patient 1.2 558815 4.439181753 12 53.27018103 Patient 1.3 588523 5.040505016 12 60.48606019 Negative 473517 2.712654497 12 32.55185396 Patient 2.1 1399455 21.45467886 12 257.4561463 Patient 2.2 1183939 17.09239313 12 205.1087176 Patient 2.3 1264326 18.71951624 12 224.6341948 Positive 1257350 18.57831417 12 222.93977 PCR Results: Summary Our positive control PCR result was 222.93977 μg/mL Our negative control PCR result was 32.55185396 μg/mL Observed results Patient 1 : The images did not fluoresce very much. Not very much green color. The initial PCR Product Concentration was 23.24660518,53.27018103, 60.48606019 respectively for each of the three trials of patient 1. Patient 2 : The images all fluoresced bright green. The initial PCR Product Concentration was 257.4561463,205.1087176, 224.6341948 respectively for each of the three trials of patient 2. Conclusions Patient 1 : Values for the initial concentration were closest to the negative control.Therefore, this patient tests negative for the diseased DNA. Patient 2 : Values for the initial concentration were closest to the positive control.Therefore, this patient tests positive for the diseased DNA.