BME100 s2014:T Group6 L5

BME 100 Spring 2014	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: Nicole D. Fisk Name: Doan-Nhi T. Tran Name: Mark A. Keppler Name: Rayan S. Altayyar Name: Brittani M. Ogden LAB 5 WRITE-UP Background Information Single-Drop Fluorimeter Device SYBR Green Dye SYBR Green Dye is a molecular dye that binds to DNA. The green dye stains double-stranded DNA as well as single-stranded DNA, but with greater preference for the former. The resulting DNA-dye-complex emits a fluorescent green light, hence "SYBR Green." Single-Drop Fluorimeter The single-drop fluorimeter is an instrument that detects the amount of fluorescent material. The spectrofluorimeter operates by shining two high-intensity light beams at the sample. How the Fluorescence Technique Works [Instructions: In your own words] Procedure Smart Phone Set Up Smart Phone Camera Settings Type of Smartphone: iPhone 5S Flash: Inactivated ISO setting: N/A White Balance: N/A Exposure: N/A Saturation: N/A Contrast: N/A Calibration The smartphone is aligned on the cradle so that the camera is focused on the illuminated drop from the side. Distance between the smart phone cradle and drop = 6.5 cm Solutions Used for Calibration Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/mL) 5.00 80 80 2.500 2.00 80 80 1.000 1.00 80 80 0.500 0.50 80 80 0.250 0.25 80 80 0.125 0.00 80 80 0.000 Placing Samples onto the Fluorimeter 1.) First, turn on the excitation light and the camera then place the camera on the cradle with adjusting the height to focus on the drop sideways. 2.) Second, place 80 microliter drop of SYBR Green 1 on the slide then place 80 microliter of the sample over the SYBR Green. 3.) Third, take a photo of the fluorimeter after lowering the lid of the box, so that it removes as much of stray light. 4.) Finally, remove the box without moving the smartphone and record the sample. Repeat for all samples. Data Analysis Sample with no DNA Sample with DNA Representative Images of Samples [Shown to the right] Image J Values for All Samples Final DNA concentration in SYBR Green I solution (µg/mL) AREA Mean Pixel Value Rawintenden of the Drop 0 56128 54.704 3070438 0.25 60188 88.251 5311680 0.5 58392 107.747 6291567 1 62152 135.245 8405724 2 75472 124.373 9386656 5 75472 201.535 15210272 Fitting a Straight Line Graph 1. PCR Results Summary Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentration of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative. Your positive control PCR result was 0.186 μg/mL Your negative control PCR result was 0.065 μg/mL Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed Patient 16946: PCR result was 0.029 μg/mL. Patient 46296: PCR result was 0.037 μg/mL. The images for both patients showed the drops as somewhat dark and opaque, with very little fluorescence. The images are similar to the sample image of a sample with no DNA shown above. Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion. Patient 16946: Negative Patient 46296: Negative