BME100 f2013:W900 Group2 L5

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OUR TEAM

Name: Leo Santos
Name: Steven Nye
Name: Robert Rye
Name: John Richards
Name: Vivian Vuong


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is used to verify the presence dsDNA in this lab. The dye fluoresces well with high concentrations of dsDNA,but fluorescence becomes less apparent as concentration decreases.


Single-Drop Fluorimeter
A single-drop fluorimeter is a device used to detect fluorescence. FluorimterVV.png
Single-Drop Fluorimeter used in this lab

How the Fluorescence Technique Works
Some SYBR Green Dye is placed on a fluorimeter slide along with a sample of concentrated dsDNA and slid into the fluorimeter. The hydrophobic Teflon coating on the slide's rough surface allows the SYBR Green Dye and dsDNA to remain a droplet on the slide for easier measurement of fluorescence instead of being absorbed into the slide. A light short in wavelength and high in energy is shined on the sample. In this case, it would be the BlueLED. The blue light excites the molecules of the SYBR Green Dye in the presence of dsDNA to fluoresce and emit a bright green fluorescence glow. The lower the concentration of dsDNA, the lesser the excitation of the molecules and more dull the fluorescence.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Android
    • Flash: No flash
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: 2.0 (Highest)
    • Saturation: No Saturation Setting
    • Contrast: No Contrast Setting


Calibration

First, the phone was placed on the cradle in front of the fluorimeter, about 8 centimeters away. Then plastic tops and other objects were used to adjust the height of the fluorimeter so that it lined up with the camera of the phone. A ruler was used to measure exactly 8 centimeters from the fluorescent light to the camera on the phone. After that, the phone was carefully focused on the droplet to provide a clear image for the analysis on ImageJ. Lastly, a black box was placed on top of the entire set-up, to block off any light.
Fluorimter SetupVV.png

Set-up of the lab

  • Distance between the smart phone cradle and drop = 8cm


Solutions Used for Calibration

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 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

  1. Place a slide, smooth side down, in the fluorimeter.
  2. Place 80µl of SYBR Green I solution in between the first and second row, in the middle circle.
  3. Make sure the use a new pipette tip every time to avoid contamination.
  4. Place 80µl of your sample solution on top of the SYBR Green I drop.
  5. Make sure to adjust the slide so that the blue fluorescent light is shining through the drop.
  6. Focus the camera on the phone and set the timer on the phone's camera.
  7. Close the cover of the box and get a picture of the fluorescence.
  8. Set the pipette to 160µl and remove the drop from the slide.
  9. Repeat the previous steps two more times for the same concentration.
  10. For every new concentration, use a new slide and repeat the same procedure.


Data Analysis

Representative Images of Samples

DNANegative.png
The picture above is a sample with no DNA (no fluorescence).


DNAPositive.png
The picture above is a sample with DNA. (fluorescence)


Image J Values for All Samples


Calf Thymus DNA concentration (FINAL), microg/ml Area Mean Pixel Value RAWINTENDEN Of the Drop RAWINTDEN Of the Background
2.5 36900 127.017 4686939 70368
2.5 36412 123.585 4499958 70857
2.5 39654 125.639 4439754 69424
1 34564 112.69 3895021 17375
1 35128 114.165 4010388 10092
1 36900 117.126 4321962 58131
0.5 38008 85.004 3230843 45942
0.5 38008 80.339 3053511 12258
0.5 40380 86.75 3502948 15361
0.25 40180 53.409 2145968 11433
0.25 44112 54.053 2384369 14314
0.25 44184 54.523 2245485 9585
0.125 48240 24.307 1172555 22813
0.125 39580 21.909 867163 15900
0.125 51344 28.452 1460840 22417
0 44112 22.333 985170 22237
0 39888 25.178 1004309 48596
0 36412 25.088 913521 53557


Fitting a Straight Line

DNAGraph.jpg