BME100 f2013:W900 Group3 L5

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OUR TEAM Error creating thumbnail: File missing Name: Marissa Kulick Error creating thumbnail: File missing Name: Blake Woods Error creating thumbnail: File missing Name: Shaun Wootten Error creating thumbnail: File missing Name: Bryce Gonzales LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green Dye is a molecular dye that is not fluorescent in water or small amounts of single strand DNA, but is very fluorescent with dsDNA (double-stranded DNA). Single-Drop Fluorimeter A Single-Drop Fluorometer is used to detect and measure fluorescence using the amount of fluorescent material, intensity, and wavelength distribution of emission spectrum. The Fluorimeter The Fluorimeter pictured above was the device used for this lab. The gap in the middle-right is for a hydrophobic slide to be inserted and then slid into position. The desired position results in the beam of light being passed through the drop of liquid. The toggle switch on the left controls the beam of light. How the Fluorescence Technique Works The fluorescence technique works by shining a light of a shorter wavelength to be absorbed by a molecule. This will excite the molecule and it will emit a brighter light with a higher wavelength. Procedure Smart Phone Camera Settings Type of Smartphone: Samsung Galaxy S1 Flash: OFF ISO setting: 150 Auto White Balance: "Auto" Exposure: "Auto" Saturation: 0 Contrast: 0 This was the only smartphone used throughout the experiment. Calibration First, we placed the smart phone in the cradle given to us, then adjusted the vertical distance by adding slides beneath the fluorimeter. After getting the height between the fluorimeter and the camera in line, we placed the phone to focus on the drop and in return capture a clear image. Error creating thumbnail: File missing ' Distance between the smart phone cradle and drop = 4.5 centimeters. Solutions Used for Calibration Calf Thymus DNA Solution Concentration (micro/mL) Volume of the 2X DNA Solution (microliter) Volume of the SYBR Green I Dye Solution (microliter) 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 Using a micro-pipetter, place 80 microliters of the SYBR Green I dye in the middle of the first row of the slide. Add 80 microliters of the desired concentration of calf-thymus solution or water to the 80 microliters of SYBR Green I dye. Make sure the solution of 160 microliters is aligned with the blue LED light! Set up the timer function on the smartphone being used, and place it into the cradle (used to avoid moving, shaking, or bumping the phone) Place the fluorometer and camera in the cradle into the light box. The light box is used to keep as much light as possible from affecting the data. Record the distance between the fluorometer and lens of the camera on the smartphone in case the apparatus must be moved before the experiment is over. Making sure the camera is focused, take 2-3 pictures of the drop on the fluorometer. Remove the light box. Use the micro-pipetter to remove the 160 microliter drop from the surface of the slide. Repeat these steps two more times for the same concentration, using different areas on the same slide. Go through these steps three times for every desired concentration, using different slides for each concentration. Data Analysis Representative Images of Samples The picture above represents the negative control, which is SYBR Green without DNA. The picture above represents the positive control, which is the SYBR Green past the point of saturation. Image J Values for All Samples ' Area Mean Min Max IntDen RawIntDen ' IntDen Concentration ug/mL 1 42076 102.625 1 255 4318055 4318055 4318055 2.50 2 42948 100.491 1 255 4315896 4315896 4315896 2.50 3 42948 105.926 4 255 4549299 4549299 4549299 2.50 4 42948 91.052 3 219 3910510 3910510 3910510 1.00 5 42948 85.141 0 255 3656651 3656651 3656651 1.00 6 42948 89.729 4 255 3853672 3853672 3853672 1.00 7 42948 60.908 0 255 2615887 2615887 2615887 0.50 8 42948 60.783 0 255 2610502 2610502 2610502 0.50 9 42948 63.747 19 255 2737802 2737802 2737802 0.50 10 42948 33.408 0 255 1434791 1434791 1434791 0.25 11 42948 33.408 0 255 1434791 1434791 1434791 0.25 12 42948 39.597 0 255 1700604 1700604 1700604 0.25 13 42948 30.104 0 249 1292917 1292917 1292917 0.125 14 42948 38.351 3 233 1647118 1647118 1647118 0.125 15 42948 34.052 0 238 1462475 1462475 1462475 0.125 16 42948 18.085 0 255 776731 776731 776731 0.00 17 42948 13.576 0 230 583066 583066 583066 0.00 18 42948 16.245 0 234 697697 697697 697697 0.00 Fitting a Straight Line The graph above establishes the relationship between the integrated density (INTDENS) readings before background correction and the calf thymus DNA concentration in micrograms per milliliter. The integrated density is the sum of independent, noninteracting subsystems that hold proportional to the amount of material in the substance paralleled in observation of the direct relationship between the integrated density and the concentration of the calf thymus DNA.