BME100 f2013:W900 Group9 L5

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OUR TEAM


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
The SYBR green dye has the ability to fluorescence when there is a presence of double-stranded DNA (dsDNA). According to Life Technologies, SYBR dyes are one of the best high-sensitivity reagents for staining DNA and RNA. The green dye binds to the DNA, causing the DNA to absorb blue light and reflect green light.

Single-Drop Fluorimeter
A single-drop fluorimeter device detects PCR-amplified DNA molecules based on the amount of flourescent material in the sample. A blue LED light is shined upon the sample; a camera can then capture green light flourescened by the DNA.
Group9 Fluorimeter.jpg

How the Fluorescence Technique Works
The fluorescence technique works by adding DNA and SYBR Green 1 dye to see how much fluorescence shines through with the picture. The higher the concentration the more fluorescence that should be visual when looking at the light shining through the drop. This happens because of the blue LED light, which shines onto the sample and excites the dye, causing it to fluorescence green light. The sample congregates at the glass spots on the slide because the rest of the slide is coated in a highly hydrophobic material.


Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: off
    • ISO setting: auto (no option on model used)
    • White Balance: auto (no option on model used)
    • Exposure: auto (no option on model used)
    • Saturation: auto (no option on model used)
    • Contrast: auto (no option on model used)


Calibration
The phone was placed in the cradle at an appropriate angle to view the slide from the side. The fluorimeter was raised in front of the phone to get the right angle (as if it phone were at eye-level with the slide). The camera was placed 7.5 cm away from the drop to ensure that the drop would be in focus. A light box was placed over the set up to provide darkness to take the picture of the light flowing through the DNA and SYBR Green 1. The fluorescence in the sample was easily seen with higher concentrations of SYBR Green 1.
Group9 CameraSetup.jpg

  • Distance between the smart phone cradle and drop = 7.5 cm

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. Attach fresh tip to micropipette and set micropipette to 50 micro-liters
  2. Push micropipette plunger to first stop, and place tip into SYBR Green 1 dye, release plunger
  3. Lift tip out of SYBR Green 1 dye and hover micropipette tip directly between two holes of the fluorimeter slide
  4. Push plunger down to second stop, allowing dye to release onto the slide
  5. Repeat steps 1-4 but now with the DNA
  6. After observing the solution on the fluorimeter, discard the solution
  7. Push micropipette plunger to first stop, place tip in the solution on the slide, and release plunger
  8. Repeat step 7 if solution still remains on slide, discard micropipette tip
  9. Repeat steps 1-8 for remaining DNA, make sure to use each hole on the slide only once


Data Analysis

Representative Images of Samples

0.0 μg/mL DNA Concentration Image J Analysis

0.0 DNA and Data.png


2.5 μg/mL DNA Concentration Image J Analysis

Group9 2.5ImageJScreenshot.jpg


Image J Values for All Samples

Calf Thymus DNA Concentration (FINAL), μg/mL Image Number AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 image 1 17828 17.006 303179 39188
2.5 image 2 20135 41.771 841061 40835
2.5 image 3 19078 45.546 868928 43759
1 image 1 20575 20.664 425158 40548
1 image 2 22096 19.014 420125 49107
1 image 3 21896 18.092 396148 46448
0.5 image 1 20792 29.294 609079 42845
0.5 image 2 21090 29.49 621944 44877
0.5 image 3 22716 29.888 678927 46969
0.25 image 1 18271 26.316 480825 35867
0.25 image 2 17540 23.916 419479 33950
0.25 image 3 17392 23.244 404253 34440
0.125 image 1 21953 13.737 301576 44788
0.125 image 2 18710 9.657 180684 41616
0.125 image 3 21953 12.31 270236 47888
0 image 1 18376 12.349 226917 40649
0 image 2 20532 10.045 206243 37931
0 image 3 17009 11.212 190697 36581


Fitting a Straight Line

Group9 CalibrationCurve.jpg