BME100 s2017:Group2 W8AM L5

BME 100 Spring 2017	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: Dallas Charles Name: Abigail Hanson Name: Daniel Jimenez Name: Vanessa Sanders Name: Kathryn Smith Name: David Walker LAB 5 WRITE-UP PCR Reaction Report The PCR pipetting experience went well because our designated pipettor had previous experience using a micropipettor. The pre-lab reading was a good refresher and helped everyone to better understand micropipetting. The difference between the first and the second stop on the pipettor was clear after some practice. The final amount of liquids in the final PCR tubes was the same, but the amount left in the PCR reaction mix and DNA sample tubes varied a little. The labeling scheme was slightly changed to fit the whole label on the tubes. Fluorimeter Procedure Imaging set-up To set up the fluorimeter, a glass slide was placed with the smooth side down in the fluorimeter and then turned on. The smartphone was placed on a stand and the height of the fluorimeter was adjusted until the camera was parallel to the side of the slide. Then, 80 microliters of the SYBR Green I solution and 80 microliters of pure water were added to the middle of the first two rows on the slide. The slide was adjusted until the solution was in line with the light of the fluorimeter. The distance of the camera to the drop of solution was adjusted until the image on the camera was clear. Finally, the light box was placed over the fluorimeter/ camera set up and the timer of the camera was turned on. This was repeated for the 5 different positions on the slide. Placing Samples onto the Fluorimeter 80 microliters of the first sample and 80 microliters of the SYBR Green I solution were micropipetted to the first position on the slide. The drop of the two solutions was aligned with the blue LED light. The light box was placed over top of the fluorimeter and phone carefully so as not to move the phone. Once the cover was placed on the light box, three focused pictures were taken of the drop. The box was then carefully removed. Next the micropipettor was used to remove the 120 microliters of solutions and the process was repeated for the rest of the samples, moving the slide to the next position. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA High (5 μg/mL) DNA sample Low (0.5 μg/mL) DNA sample Zero DNA sample 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 18767242 18007510 18520300 18431684.0 387540.7 2 1 C-2 17841400 18524395 25449362 20605052.3 4209171.3 1 0.5 C-3 20741660 19339816 19927087 20002854.3 703986.6 0.5 0.25 C-4 6683757 7279467 7324531 7095918.3 357652.6 0.25 0.125 C-5 14701120 12438577 13170951 13436882.7 1154476.0 0 0 C-6 -76562 -67442 -59502 -67835.3 8536.8 Calibration curves Images of Our PCR Negative and Positive Controls Negative Control PCR Sample Positive Control PCR Sample PCR Results: PCR concentrations solved PCR Product TUBE LABEL MEAN PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration (µg /mL) G2 + 18466411.3 0.623320567 12 7.48 G2 - 7760420.0 0.088021 12 1.06 G2 1-1 21484117.7 0.774205883 12 9.29 G2 1-2 16955910.0 0.5477955 12 6.57 G2 1-3 16351957.7 0.517597883 12 6.21 G2 2-1 5846370.0 -0.0076815 12 -0.09 G2 2-2 3405786.0 -0.1297107 12 -1.56 G2 2-3 6698147.3 0.034907367 12 0.42 PCR Results: Summary Our positive control PCR result was 7.48 μg/mL Our negative control PCR result was 1.06 μg/mL Observed results Patient 75263 : The drops for patient 75263 all glowed under the florescent light. The results for patient 75263 gave us 9.29 μg/mL, 6.57 μg/mL, 6.21 μg/mL for the three trials, giving us an average of 7.36 μg/mL for patient 1. Patient 45868 : The drops for patient 45868 gave us -0.09 μg/mL, -1.56 μg/mL, and 0.42 μg/mL for the three trials which gives an average of -0.41 μg/mL for patient 2. Conclusions Patient 75263 : Patient 75263's results and average are much closer to the positive control. Because of this we believe the patient has SNP. Patient 45868 : Patient 45868's results and average are much closer to the negative control leading to the conclusion that this patient does not have SNP. Extra Credit: Electrophoresis Gel The numbers on the electrophesis gel image correspond to which DNA sample was put into each well. The samples were marked and prepared for the electrophesis in the previous lab and the numbers on the image correspond to the PCR Reaction samples of the two patient samples received during that lab. Well 1 contained G2 P, the positive control and Well 2 contained G2 N, which was the negative control. Wells 3-8 contained the reaction samples of Patient 1 and 2 and three replicas for each. That being said, Well 3 contained G2 1-1, patient 1, replica 1, Well 4 contained G2 1-2, patient 1, replica 2, Well 5 contained G2 1-3, patient 1, replica 3. The samples for patient 2 started at Well 6 which contained G2 2-1, patient 2, replica 1, and continued with Well 7 which contained G2 2-2, patient 2, replica 2, and Well 8 contained G2 2-3, patient 2, replica 3. The last well, Well 9, contained the ladder, which helped to compare the 8 true PCR samples.