BME100 f2013:W900 Group4 L5

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OUR TEAM

Name: Elliott Tejada
Name: Sierra Headrick
Name: Philip Terzic
Name: Aaron Blank


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
The SYBR Green Dye is a special florescent dye that was used to help sense the presence of an enzyme in a DNA sample. The dye is dependent on how strong the enzyme is to make the sample glow a bright green.


Single-Drop Fluorimeter
A Single-Drop Fluorimeter is a structure inside of another structure. The outer layer of the device is a shield like structure that is designed to keep the light out. The structure that is placed on the inside is composed of two components, a phone holder and the actual fluorimeter. The fluorimeter has a slot that allows for a glass slide with small indentations, referred to by the group as craters, to hold the drops in place upon shutting the device and capturing a picture of the DNA sample. There is a black light that makes the fluorescent enzyme show up as a green color instead of a clear blue.
SingledropfluorimeterBME100.jpg


How the Fluorescence Technique Works
The technique used for the fluorescence is brought out when the correct enzyme is in the DNA sample. For a clearer picture of the sample, the box was shut to enhance the quality of the photograph. A phone was place on the bench with the flash off and the timer was set to 10 seconds to have time to focus and sharpen for a better picture. Three pictures were taken for each sample to ensure that there was a clear picture. It was repeated for each sample 3 times, but on the next section of craters of the glass plate.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5s
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: N/A
    • Saturation: Auto
    • Contrast: Auto


Calibration
The smartphone was placed in the cradle and adjusted so that it was positioned vertically from the slides beneath the fluorimeter. Then, the distance between the drop and the iPhone5s was measured and the camera was set to focus on the position of the drop. The lid was closed to block out as much possible light, while still allowing access to the camera button. Each drop was photographed three times to provide a wider selection of data and to correct for any outliers. Three images from each concentration were then analyzed using ImageJ by splitting the color channels and selecting the green color scale, drawing a precise oval around the drop and then measuring area, mean pixel value and the integrated density for the drop and the background. The resulting value determined by the following equation, Integrated Density of Drop-Integrated Density of Background, was plotted versus the concentration. The linear best fit line of this plot was then found using Excel's Best Fit function. This best fit line can be used in future measurements to plug in the concentration being used for the value "x" and to determine the calibrated integrated density "y".

  • Distance between the smart phone cradle and drop = 4.2cm


Solutions Used for Calibration

Calf Thymus DNA Solution Concentration (microg/ml) Volume of the 2X DNA solution (uL) Volume of SYBR Green I Dye Solution (uL) Final Concentration SYBR Green assay (ng/ml)
5 80 80 2.5
2 80 80 1
1 80 80 .5
.5 80 80 .25
.25 80 80 .125
0 80 80 blank


Placing Samples onto the Fluorimeter

  1. First, use the plastic pipette to place 80 micro liters of SYBR green onto a middle dot on the glass slide in the fluorimeter.
  2. Dispose of used pipette tip.
  3. Using the plastic pipette, place 80 micro liters of the sample solution onto the same dot the SYBR green occupies.
  4. Next, adjust the fluorimeter so the blue led light is focused on the liquid drop.
  5. Place the camera phone 4.2 cm from the fluorimeter.
  6. Cover the fluorimeter with the light box.
  7. Use the timer on the camera phone to take two pictures of the sample. Make sure the camera is focused before the images are taken.
  8. Use the plastic pipette to remove the drop from the glass slide.
  9. Repeat the above method three times for each concentration being evaluated. Make sure to change out pipette tips between each new use.


Data Analysis

Representative Images of Samples

Sample Images Prior to Being Imported and Analyzed in ImageJ:
FlourometerSamples.jpg

Flourometer Sample with No DNA (0 μg/mL) Loaded into ImageJ, Green Portion with Droplet Selected
NoDNASample.jpg

Flourometer Sample with Positive DNA (2.5 μg/mL) Loaded into ImageJ, Green Portion with Droplet Selected
2.5DNASample.jpg


Image J Values for All Samples

Calf Thymus DNA Concentration (FINAL), μg/mL Image Number per Concentration AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN - BACKGROUND
2.5 image 1 119996 11.256 13350307 53547 13296760
2.5 image 2 125354 133.743 16765215 108685 16656530
2.5 image 3 95556 107.189 10242528 124282 10118246
1 image 1 107614 73.834 7945597 393837 7551760
1 image 2 102803 82.679 8499654 110354 8389300
1 image 3 99527 77.862 7749333 265831 7483502
0.5 image 1 112252 60.301 6768935 105892 6663043
0.5 image 2 89572 55.755 4994050 158289 4835761
0.5 image 3 83212 72.921 6067889 84288 5983601
0.25 image 1 91640 59.384 5441952 121697 5320255
0.25 image 2 82956 72.513 6015402 74851 5940551
0.25 image 3 92852 60.59 5625901 82502 5543399
0.125 image 1 122868 33.051 4060960 1047687 3013273
0.125 image 2 110620 25.781 2851901 845335 2006566
0.125 image 3 135620 3.054 5009564 414250 4595314
0 image 1 54732 80.432 4402189 113462 4288727
0 image 2 83472 11.407 952169 837 951332
0 image 3 106368 36.909 3925957 1411528 2514429


Fitting a Straight Line
ImageJPlotResize.jpg