BME100 f2013:W900 Group9 L5

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BME 100 Fall 2013 Home
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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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.

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.


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)

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.

  • 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

2.5 μg/mL DNA Concentration Image J Analysis

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
2.5image 11782817.00630317939188
2.5image 22013541.77184106140835
2.5image 31907845.54686892843759
1image 12057520.66442515840548
1image 22209619.01442012549107
1image 32189618.09239614846448
0.5image 12079229.29460907942845
0.5image 22109029.4962194444877
0.5image 32271629.88867892746969
0.25image 11827126.31648082535867
0.25image 21754023.91641947933950
0.25image 31739223.24440425334440
0.125image 12195313.73730157644788
0.125image 2187109.65718068441616
0.125image 32195312.3127023647888
0image 11837612.34922691740649
0image 22053210.04520624337931
0image 31700911.21219069736581

Fitting a Straight Line

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