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 A polymerase chain reaction (PCR) was utilized to amplify the DNA from 2 patients identified here as 46296 and 16946. A positive and negative control were amplified alongside the patient’s DNA. PCR results were analyzed by using SYBR Green and a blue light emitting diode (LED) capable of causing the SYBR to fluoresce if DNA was present in the sample. Before the patient samples could be analyzed, known concentrations of calf thymus DNA had to be used in conjunction with the SYBR to create a curve fit correlating the calf thymus DNA concentration to the intensity of fluorescence. An iPhone 5 was used to take snap shots of 160 μL droplets, consisting of 80 μL of calf thymus solution that ranged from 0 – 5 μg/mL and 80 μL of SYBR Green. The photos were imported into Image J, a public, java based program available through the National Institute of Health (NIH). Image J has a function that calculates the integrated pixel density of image samples. The integrated density (RAWINTDEN) of the samples and the known DNA concentrations were incorporated in Graph 1 above.