BME100 f2018:Group4 T1030 L5

BME 100 Fall 2018	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 Oscar Rodriguez Nadia Altamirano Abigail Turcheck Alejandra Solis Leah Woods LAB 5 WRITE-UP PCR Reaction Report The objective of PCR lab 3 was to prepare DNA samples to be placed in the thermal cycler to conduct the PCR process. The materials required was the DNA samples from two different patient’s, a pipette, disposable pipette tips, PCR tubes, positive and negative controls, and eight tubes of primer solution to mix with the patient samples and the controls. First, the empty PCR tubes were separated into sections of four connected PCR tubes and Leah and Abby labeled with the patient’s information. While the PCR tubes were being labeled Nadia, Alejandra, and Oscar reviewed the lab workbook for the correct techniques to use while combining the multiple components for PCR. Abby used the micropipette to first transfer 50 microliters of PCR reaction solution to the empty PCR tube. After discarding the pipette tip after the first transfer, Abby then transferred the 50 microliters of primer mix into the same PCR tube to make a total of 100 microliters. This process was repeated with the other seven PCR tubes with the end result being one positive and one negative control of PCR solution, three tubes containing the DNA of patient one, and three tubes that contained the DNA of patient two. Once all tubes had the DNA and the primers, the PCR tubes were placed in the thermal cycler to conduct the continuous heating and cooling process. All members of group four properly discarded the pipet tips and the supplies used to transfer the controls, patient replicates, and the DNA primer mix. Fluorimeter Procedure Imaging set-up The fluorimeter was placed upon a platform so that the smartphone camera would be aligned to take a picture from the side. The smartphone camera was set to take three photos with no flash on a five-second countdown timer so that the lightbox could be closed in time. When ready, the lightbox was placed over the fluorimeter so that when closed, a minimal amount of light could shine through the door and accurate photos could be taken of the sample. Placing Samples onto the Fluorimeter Step 1: An 80 μL drop of SYBR GREEN solution was placed upon the glass slide near the front, in between the two right rows so that the drop was round and stabilized. Step 2: 80 μL of the sample solution was added to the drop of SYBR GREEN, making sure the full drop was stabilized in between the two dots. Step 3: The glass slide was moved so that the blue LED light was focused through the solution drop onto the black fiber optic on the left side of the fluorimeter. Step 4: A light box was placed over the fluorimeter. The smartphone was placed on the cradle so that the side of the drop was in focus. A timer was set to take three photos, and the light box was closed for this time. Step 5: All steps were repeated for all concentrations of calf thymus DNA and the PCR samples. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA 5 micrograms/mL Calf Thymus DNA .5 micrograms/mL Calf Thymus DNA 0 micrograms/mL Calf Thymus DNA (pure water) 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 16422500 14444505 12995204 14620736.33 1720430.929 2 1 C-2 14663491 13546174 12172894 13460853 1247488.721 1 0.5 C-3 12075274 12801666 12643553 12506831 382009.1797 0.5 0.25 C-4 7734444 8847778 8855907 8479376.333 645143.1284 0.25 0.125 C-5 3867222 4753889 4427953.5 4349688.167 448484.8816 0 0 C-6 649631 421687 1566481 879266.3333 605960.0774 Calibration curves Plot 1, comparing calf thymus DNA concentrations and the MEAN RAWINTDEN - BACKGROUND value Plot 2, comparing calf thymus DNA concentrations and the MEAN RAWINTDEN - BACKGROUND value without the highest concentration value 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 RAWINTDEN DROP - BACKGROUND Mean Standard Deviation Image 1 Image 2 Image 3 G4-neg 627331 616586 618780 620899 5677.268445 G4-pos 729022 752644 725177 735614.3333 14872.9004 G4-1 (1) 10177169 9705804 10150462 10011145 264770.0147 G4-1 (2) 3223111 3305722 3139767 3222866.667 82977.7698 G4-1 (3) 16391910 15389034 15237078 15672674 627493.4057 G4-2(1) 8727631 8384113 8877497 8663080.333 252946.7025 G4-2 (2) 7786897 6731392 7217747 7245345.333 528293.4339 G4-2 (3) 4769311 4908953 4269998 4649420.667 335925.7984 PCR Results: Summary Our positive control PCR result was -4.529 μg/mL Our negative control PCR result was -4.758 μg/mL Observed results Patient 46547 : The images showed high levels of green fluorescence. The concentration levels found for the three samples (in μg/mL) were 14.022, .446, and 25.35. Patient 41967 : None of the images exhibited any green fluorescence. The concentration levels found for the three samples (in μg/mL) were 11.32, 8.49, and 3.30. Conclusions Patient 46547 Positive, due to fluorescence observed and higher mean concentration value (like the positive control). Patient 41967 Negative, due to lack of fluorescence and lower mean concentration value (like negative control).