BME100 f2015:Group8 8amL5

BME 100 Fall 2015	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: Riley Barnett Name: Manuela Hiche Schwarzhaupt Name: Nicholas Grant Name: Chris A Pina 1 Name: Cody Wong Name: Luis Montero LAB 5 WRITE-UP PCR Reaction Report Our experience with micro pipetting was rewarding. The tutorials and pre lab assignments allowed us to prepare for this lab and we were able to comply to each of the procedural steps associated with micro pipetting. We were able to understand the difference between the first and second stop on the pipette allowing us to avoid problems or inconsistencies in our mixtures. Each of the final reactions have the same amount of liquid and there was no remaining liquid in the tubs where we acquired the DNA and PCR mixture. We did not have to change the labeling of our tubes. We labeled our tubes with a black marker on the side of the tub. In total we were able to enjoy ourselves during the lab and we were able to utilize what we had learned to properly us a micro pipette. Fluorimeter Procedure Smart Phone Camera Settings Type of Smartphone: LG G4 Flash: off ISO setting: 2700 White Balance: Automatic Exposure: High Saturation: High Contrast: Low Camera set-up Distance between the smart phone cradle and drop = 5 cm Placing Samples onto the Fluorimeter Wear gloves and a lab coat. Place camera with pre set settings on cradle and set the timer to 3 seconds. Use a plastic tray to adjust the fluorimeter so the camera lens faces the drop face on. Place a slide inside the fluorimeter with the rough part of the glass pointing up. Using a ruler to record the distance between the camera and the first 2 rows of the slide inside the fluorimeter to a close distance of 4 cm or greater. Turn on the switch for the Blue LED light. Using a mircopipetter, place 80μL of the SYBR GREEN I solution in the first 2 rows of the slide. Place 80 μL on one of the calf thymus solutions provided on top of the SYBR GREEN drop to create 1 sample drop. Make sure that the slide is adjusted, so the Blue LED light goes through the center of the sample drop on to the other side. Take three images of the sample drop making sure it is focused and that the light box covers as much light as possible on each take. Carefully remove the box without moving the smartphone of set up. If any movement occurs, make sure to set it up to the original distance. Remove the sample drop using the micropipetter and discard of the sample in the appropriate container. Repeat steps 5- 10 for the remaining calf thymus solutions provided, making sure to either move the slide to a new set of rows or use a brand new slide. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Image of High Calf Thymus DNA Image of Low Calf Thymus DNA Image of Zero Calf Thymus 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 19766016 16456142 19258756 18493638 1782658 2 1 C-2 11412240 9067981 8465690 9648637 1556731 1 0.5 C-3 13400822 14203955 13347413 13650730 479851 0.5 0.25 C-4 8226707 8030317 7741956 7999660 243825 0.25 0.125 C-5 5211504 7920480 5968735 6366906 1397692 0 0 C-6 1467917 2368149 2505492 2113853 563596 Calibration curves Images of Our PCR Negative and Positive Controls PCR Positive Control PCR Negative 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)" G8 + 53719487 76199145 12 914389740 G8 - 22094584 23490973 12 281891680 G8 1-1 19099824 18499707 12 221996480 G8 1-2 40015513 53359188 12 640310260 G8 1-3 31971056 39951760 12 479421120 G8 2-1 37814774 49691290 12 596295480 G8 2-2 41920503 56534172 12 678410060 G8 2-3 38059206 50098677 12 601184120 PCR Results: Summary Our positive control PCR result was 914389740 μg/mL Our negative control PCR result was 281891680 μg/mL Observed results Patient 27435 : The droplet did not have any visible green. ImeageJ was not able to see as much SYBR green when analyzing the pictures of the droplets. The average SYBR green that was exhibited in the samples was 447242620 μg/mL Patient 16764 : The droplet did not have any visible green. ImageJ was able to analyze the droplet and showed that there was a minute amount of green inside the droplet that we did not initially see. The average of SYBR green was 625296553.3 μg/mL Conclusions Patient 27435 : Negative Patient 16764 : Positive We found that based on the results of the PCR product concentrations that patient 16764 was Positive and patient 27435 was Negative. We came to this conclusion by comparing the PCR product concentrations with that of the Positive and Negative controls. Two out of the three samples were negative and therefore we concluded that the patient was negative. Patient 27435 was three out of three for the Positive control and came to the conclusion that the patient was indeed positive.