BME103 s2013:T900 Group3 L2

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Contents

OUR TEAM

Jonus Reyna: Data Analysis
Jonus Reyna: Data Analysis
Amnah Alkhan: Procedure
Amnah Alkhan: Procedure
Haifa Takrooni: Background
Haifa Takrooni: Background
Name: student
Name: student
Name: student
Name: student
Name: student
Name: student


LAB 2 WRITE-UP

Background Information

References:http://en.wikipedia.org/wiki/SYBR_Green_I

SYBR Green Dye

SYBR Green (cyanine family) is a small molecular dye used in different scientific fields as biochemistry and molecular biology. One of SYBER Green function is to satin the DNA ( deoxyribose nucleic acid) double stranded. This dye fluorescence well and strongly with DNA double-stranded chain under blue ray in single- Drop Fulorimeter device to emits green light , but weakly with water and single stranded of DNA.


Single-Drop Fluorimeter

is a device that designed for a reason which is to look for presence of DNA double stranded by measuring its fluorescence for green light that is result due to detect pieces of the double strand DNA.


How the Fluorescence Technique Works

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The process of the fluorescence technique based on having different variables which are :a hydrophobic properties (water-hating) surface. This was used in order to avoid absorbing of water drops during the experiment. A slide top coated with rough adhesive Teflon layer to keep the molecules n place and spherical shaped during analysis.That allow 2 important things to occur which are the blue LED ray to be focused in the drop; in order to increase intensity of the molecular and stimulate the fluorescent environment. Also, adding of SYBER Green with a solvent helped form a complex with DNA that preferred to go to the surface rather than anywhere else on the Fluorimeter. However, If a water-soluble dye was used instead of DNA dye the outcome of the experiment would be different because instead of causing green glow light it reflect light off of the drop.

Procedure

Samsung Galaxy S3:

Flash: off

ISO sitting: Auto

White balance sitting: Auto

Exposure Value: 0

Contrast: off


Calibration

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A good distance and angle between the smart phone and the drop helped to get better results. The smartphone was placed at the best distance that allowed the camera to focus on the first two rows of the slide. Plastic objects were used to raise the base of the slide holder. This allowed the camera to snap photos of the drops at side angles.

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


Solutions Used for Calibration

Calf Thymus DNA solution (microg/mL) Volume of DNA Solution (μL) Volume of SYBR GREEN I Dye solution (μL) Final DNA concentration in PicoGreen Assay (ng/mL)
0 80 80 blank
.25 80 80 .125
.5 80 80 .25
1 80 80 .5
2 80 80 1
5 80 80 2.5


Placing Samples onto the Fluorimeter

1- Measure the best distance and angle between the smartphone and fluorimeter. (first two rows)

2- Place the hydrophobic slide into the machine and single-drop fluorimeter. Make sure that the Blue light crosses the first two rows.

3- Use the plastic pipette to transfer an 80-microliter drop of Syber Green from the Microcentrifuge tube to the first two rows on the hydrophobic slide. Then add an 80-microliter of calf thymus solution to the same drop.

4- Use a box to isolate the smartphone and the fluorimeter from the light as much as it possible, then while take pictures of the drop.

5- Set the timer on the smartphone’s camera, then take a picture after closing the box and cover all sides.

6- Take three pictures of the drop inside the box without moving the smartphone or changing the distance.

7- Remove the box. Make sure not to move the smartphone to maintain its distance from the box. If the smartphone is accidentally moved, use a ruler measure and place the smartphone at the same distance that was measured in step 1.

8- To remove the 160-microliter drop, use the pipette. Move the slide forward until the Blue light cross the 3rd and 4th rows instead of the first two rows for next drop. (each drop needs different rows. So, you might need more than one slide)

9- For each sample “drop” of the same concentration of DNA, take three different measurements.



Data Analysis

Representative Images of Samples

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Image J Values for All Samples

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Fitting a Straight Line

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