BME100 f2013:W1200 Group1 L5

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Contents

OUR TEAM

Name: Chloe Kwon
Name: Chloe Kwon
Name: Jeff Miles
Name: Jeff Miles
Name: Casey Weinstein
Name: Casey Weinstein
Name: Nima Afzalian
Name: Nima Afzalian
Name: Mario Gonzalez
Name: Mario Gonzalez


LAB 5 WRITE UP

Background Information

SYBR Green Dye
SYBR Green Dye is a small molecular dye that does not fluoresce well in water or with single strands of DNA, but fluoresces very well with dsDNA. By combination with the DNA solution, SYBR emits a green light and produce a strong fluorescence.


Single-Drop Fluorimeter
The single-drop fluorimeter detects fluorescence within the drop of DNA solution. It measures the wavelength distribution, and intensity when light passes through the DNA solution.


How the Fluorescence Technique Works
The fluorescence technique works by combining a fluorescent SYBR green dye with the DNA solution. When the blue light beam from the single-drop fluorimeter, the drop will turn a fluorescent green color, indicating the presence of DNA. The droplet's color will be detected by the smartphone and the picture will then be analyzed using the ImageJ software.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: HTC One
    • Flash: off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Neutral
    • Saturation: Neutral
    • Contrast: Lowest Setting


Calibration

1. Switch the blue light on on the fluorimeter.

2. Adjust camera settings on smart phone to match the above settings.

3. Place smart phone in cradle so that it is perpendicular to the table and the camera lens is level with the glass slide and the drop.

4. Place cradle and phone as close as possible to the drop so that the drop remains in sharp focus.

5. Use the provided ruler to measure the distance between the drop and the camera lens. In our case, this distance was 8.5 cm.

  • Distance between the smart phone cradle and drop = 8.5 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. Insert the slide into the fluorimeter, smooth side down.

2. Use the micropipette to place 80µL of the SYBR green dye in the second row of the middle column of the slide.

3. Micropipette 80µL of the specific calf thymus DNA on top of the SYBR green dye.

4. Ensure that the light is turned on. Then start self timer on phone and place light box over entire set up.


Data Analysis

Representative Images of Samples

No DNA

With DNA


Image J Values for All Samples

Image:group1imagejdata.jpg


Fitting a Straight Line

Image:group1imagejgraph.jpg



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