BME100 f2013:W900 Group13 L5

BME 100 Fall 2013	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: Tyler Angell Name: Reem Gerais Name: Andrew Luc Name: Allison Marley Name: Cory Riecken LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green Dye is a reagent used to stain DNA. After combining the SYBR Green dye with a double stranded DNA (Single stranded DNA works as well, but produces less effective results), the solution will be able to absorb blue light and emit green light. By measuring the green light, one will be able to measure how much DNA is present in the solution. Single-Drop Fluorimeter The single-drop fluorimeter device is a box that emits a blue light. A slot is present on the device for a glass slide to be placed on. Droplets of the SYBR Green dye and DNA solutions are placed on the glass slide for the light to be passed through. How the Fluorescence Technique Works The fluorescence technique works by combining a fluorescent dye to the DNA. In this lab, SYBR Green dye was used as the fluorescent dye. The droplets will remain in place due to the Teflon coated glass. A beam of blue light is emitted from the single-drop fluorimeter and is passed through the droplet. A fluorescent green light will be emitted from the droplet which will be measured by taking a picture on a smartphone and analyzing the picture file on ImageJ. Procedure Smart Phone Camera Settings Phone: Galaxy S3 Flash: Off ISO setting: 800 White Balance: Auto Exposure: Maximum Saturation: Maximum Contrast: Minimum Calibration 1. Adjust the camera settings on the smart phone so that they match the settings above. 2. Place smart phone in the cradle. 3. Make sure that the distance from the smart phone and the cradle is 11 cm. Solutions Used for Calibration Calf Thymus DNA Concentration (Microg/ml) Volume of the 2X DNA Solution (ul) Volume of the SYBR GREEN I Assay (ng/mL) Final DNA Concentration in SYBR GREEN I Assay (ng/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 blank Placing Samples onto the Fluorimeter Before the sample can be placed on the Fluorimeter, the fluorimeter must be set up. This is done by first by placing the glass down on the fluorimeter; the glass must be placed so that the rough side is faced up. The fluorimeter must be turned on. Adjust the glass slide so that the blue light shines in the center of two rows. The hieght of the fluorimeter may need to be adjusted so that the camera has a clear view of the light. For the next steps, the micropipet will be needed. Begin by adjusting the volume on the micropipet to 80μL, and placing a new micropipet tip on the micropipet. Use the micropipet to gather 80μL of the SYBR Green I to be placed on the slide. The SYBR Green I will be placed on the glass slide for each sample. Be sure that the camera settings are all in place and take photograph of solely SYBR Green I, this is your blank. Next, use the micropipet to gather 80μL of the first sample, 0.25, and place it into the 80μL SYBR Green I present on the slide. Be sure that the light from the fluorimeter is in line with the droplet. Photograph the sample and SYBR Green I. Remove sample from slide with micropipet and repeat steps for each sample. After all samples have been collected (0,0.25,0.5,1,2,5) repeated this process two more times for a total of three pictures per sample. Data Analysis Representative Images of Samples Sample with no DNA Sample with DNA Image J Values for All Samples Calf Thymus DNA Concentration (Final), Microg/ml AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND Trial One Blank 23332 25.472 544298 544298 0.125 36784 82.709 3042371 3042371 0.25 24824 66.066 1640022 1640022 0.5 21572 67.4 138451 138451 1 21796 97.793 2151322 2151322 2.5 22144 113.478 2512864 2512864 Trial Two Blank 22172 30.492 676054 676054 0.125 22986 16.12 370541 370541 0.25 23060 74.497 1728268 1728268 0.5 26336 76.749 2021273 2021273 1 23734 126.199 2995195 2995195 2.5 23589 175.595 4142091 4142091 Trial Three Blank 24210 15.191 367788 367788 0.125 21472 10.899 134023 134023 0.25 21530 61.485 1323755 1323755 0.5 25448 95.74 2436374 2436374 1 27404 119.256 3268082 3268082 2.5 26342 131.337 3459687 3459687 Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]