BME103 s2013:T900 Group9 L2

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Coley White Name: Aimen Vanood Name: Brady Falk Name: Vignesh Senthil LAB 2 WRITE-UP Background Information Photo of the Single-Drop Fluorimeter Device. (Image used from Google Images, http://openwetware.org/wiki/BME103:T130_Group_6) SYBR Green Dye SYBR Green Dye is a type of dye that is used to highlight when dsDNA is present because that is when it fluoresces the strongest. The dye fluoresces weak with water or single strands of DNA. Single-Drop Fluorimeter The Single-Drop Fluorimeter (as pictured above) is a custom device designed for the purpose of finding when a double-stranded piece of DNA is present. It fluoresces double-stranded DNA fragments in a green color, and does nothing to a solution without the double-stranded piece of DNA. How the Fluorescence Technique Works The process had several different variables that were chosen for very specific reasons. The slide to be used was chosen because it had a rough surface with glass circles exposed in it, causing the solution to concentrate at those locations and be easier to examine. The DNA dye chosen for the experiment was used because it was not completely soluble, which allowed for the light to glow instead of causing a reflective light off of the drop. Procedure Smart Phone Camera Settings Type of smart phone used and adjusted camera settings. Phone: HTC Droid Incredible 2 Flash: None ISO setting: 800 White Balance: Auto Exposure: +2 Saturation: +2 Contrast:-2 Calibration Photo of how camera is pointing at, and focusing in on, the drop. (Image used from Google Images, http://openwetware.org/wiki/BME103:T130_Group_9) The phone was set up in the phone stand so that the camera was focusing on the drop. It was zoomed in far enough to notice the color of the drop and how it changed with each concentraton change. Distance between the smart phone cradle and drop = 3.7 cm Solutions Used for Calibration [See worksheet page 5] Image J Values for All Samples [See worksheet page 5] Calf Thymus DNA solution concentration (microg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in PicoGreen 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 Materials SYBR green Pipette Water Hydrophobic Tray Smartphone Cradle Box (For Cover) Plastic Base Step 1: Calibrate your smartphone to ensure the proper imaging environment. Step 2: Place the pre-determined concentration droplet of SYBR green onto the hydrophobic tray between the initial 2 ruts. Step 3: Make sure the droplet is centered on the green light to ensure proper imaging. Step 4: Place the hydrophobic tray onto the cradle. Step 5: Set up the smartphone on the plastic base a fixed distance away from the hydrophobic tray. Step 6: Level the view of the camera to show the droplet in a horizontal perspective. Step 7: Place the box over the contraption to create a dark room. Step 8: Take the picture of the droplet in the closed environment. Step 9: Repeat the initial steps for all the sample trials. Step 10: Use the pictures to analyze the affect of the concentration of SYBR green in the droplet samples. Data Analysis Representative Images of Samples Sample with no DNA Sample with DNA (positive signal) Calf Thymus DNA concentration (FINAL), migrog/mL Area Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the background Water 40636 32.437 1318101 302462 0.25 39770 64.565 2567761 243302 0.25 43440 48.047 2087153 50662 0.25 38994 82.158 3203667 239145 0.5 45724 105.339 4770813 286980 0.5 37690 83.846 3160168 251141 0.5 38724 73.079 2829899 54829 1.00 41200 105.656 4353036 288678 1.00 43584 113.023 4926005 335726 1.00 41404 105.037 4348994 279375 2.00 40024 135.205 5411434 326434 2.00 40968 103.250 4229944 260408 2.00 40111 129.871 5209254 425061 5.00 40950 132.782 5437418 393246 5.00 40468 147.876 5984250 352233 5.00 39796 140.267 5582067 341520 Fitting a Straight Line