BME100 f2015:Group1 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: Tiffany Nguyen Name: Maria Dooling Name: Brenden Smith Name: Nolverto Guiterrez Name: Dalia Sarbolandi Name: Nathaniel Olsen LAB 5 WRITE-UP PCR Reaction Report The experience with micro pipetting taught us a lot. The pre-lab assignments as well as the tutorials online prepared us well for this lab. They gave us experience with how to work a micro pipetter with confidence. Furthermore, they clearly explained the differences between the first and second stop on the pipette. This allowed us to avoid inconsistencies in our data. It was particularly important in this lab to be able to correctly work a micro pipetter. Our final reactions had an equal amount of liquid so there was not remaining liquid in the DNA and PCR mixture. The tubes were labeled with marker. The tutorials and pre lab materials helped accomplish this. All in all, much was learned about the process of micro pipetting and how to use a fluorimeter. Fluorimeter Procedure Smart Phone Camera Settings Type of Smartphone: Iphone 5 and Iphone 6 Flash: Off ISO setting: NA White Balance: NA Exposure: High Saturation: High Contrast: Low Camera set-up Our camera was placed into the cradle and lifted Distance between the smart phone cradle and drop = 5.5 cm Placing Samples onto the Fluorimeter/Taking Fluorimeter Measurements Put on gloves and find the smooth side of the glass slide Turn on the fluorimeter Place slide in the fluorimeter smooth side down Set cellphone camera for 3 seconds and place in the cradle Adjust height of fluorimeter so that the camera will take a picture of the slide edge-on Place an 80uL drop of the SYBR Green I solution on the first two clear circles in the middle of the slide Place an 80uL drop of the sample/calibration solution directly on top of the SYBR Green I solution Move the slide around until the light hits the drop and focuses the light on the other side Adjust the distance between the smartphone and the fluorimeter so that is is greater than 4cm away but not blurry. Record set distance. Cover with lightbox and check that the camera is focused Press camera button and lower flap before picture it taken Discard liquid drop Move slide to the next position Repeat steps 1-14 with all 5 possible measurement positions. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA High Calf Thymus DNA Low Calf Thymus DNA Zero Calf Thymus DNA Calibrator Mean Values Initial Concentration of 2x Calf Thymus DNA solution (µg/mL) Final DNA Concentration in SYBR Green I solution (µg/mL) Sample # RAWINTDEN Drop - Background Mean Std Dev Img 1 Img 2 Img 3 5 2.5 G-1 18678998 17876541 19128456 18561332 634198 2.5 1.25 G-2 12562821 13982346 11287635 12610934 13480000 1 .5 G-3 11783472 10236814 9126734 10382340 1334334 .5 .25 G-4 8345724 8178942 7976254 8166973 185026 .25 .125 G-5 5422637 6134876 5798234 5785249 356297 0 0 G-6 2634578 2390456 2623987 2549674 137988 Calibration curves Images of Our PCR Negative and Positive Controls Postive 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)" G1+ 9254872 1.2134 12 14.5608 G1- 4325825 5.5772 12 66.9264 G1 1-1 3924573 5.8829 12 70.5948 G1 1-2 4182736 5.7621 12 69.1452 G1 1-3 4623549 5.2734 12 63.2808 G1 1-4 3789251 6.2473 12 74.9676 G1 1-5 4489745 5.4905 12 65.886 G1 1-6 4872369 4.9235 12 59.082 PCR Results: Summary Our positive control PCR result was 14.5608 μg/mL Our negative control PCR result was 66.9264 μg/mL Observed results Patient 31801: After combining the SYBR green and the patient's PCR sample, it was evident that the patient was negative. No visible sign of green was detected through the fluorimeter or ImageJ. Patient 21190 : The second patient also came up as negative after the test. The fluorimeter and ImageJ did not detect any sign of green within the sample. Conclusions Patient 31801 : Negative Patient 21190 : Negative Based on the results of the PCR product concentrations, we found that patients 31801 and patient 21190 were both negative for the disease. No green appeared in the pictures from the fluorimter in either patient so this is how the conclusion was reached that both patients were negative.