BME100 f2013:W1200 Group8 L5

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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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OUR TEAM

Name: Colby Mark - Analysis Specialist
Name: Andrew Wills - Experimental Specialist
Name: Madison Grayson - Data and Procedure Specialist
Name: Hayden McIver - Experimental Specialist
Name: Isai Valdez - Research Specialist


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
Is a small molecular dye that fluoresces very well in the presence of dsDNA but fluoresces very weakly in water or with single strands of DNA. The dye binds to DNA which then absorbs blue light and emits green light.


Single-Drop Fluorimeter
Is an instrument that detects fluorescence, quantity is related to the amount of florescent material and, less directly, proportional to the amount of the molecule being detected.
Flour.jpg

How the Fluorescence Technique Works
The Fluorescence technique invovles adding dye that attaches to the DNA, which when you put blue light through it, excites the molecules causing an emission of light. This then shows the concentration of DNA in the sample.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: HTC One
    • Flash: off
    • ISO setting: 800
    • White Balance:Auto
    • Exposure:High
    • Saturation:default
    • Contrast:default


Calibration

The camera of the HTC One phone was lined up level with the fluorimeter and the flash was turned off. A photo of this can be seen in the above introduction section. The phone was placed in a vertical position on the cradle. The test strip was elevated to match the height of the phone in the cradle.

  • Distance between the smart phone cradle and drop = The distance between the smart phone cradle and the drop had to be a minimum of 4 centimeters. Our distance was exactly 7 centimeters for every picture.


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 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


Placing Samples onto the Fluorimeter
Step 1- Place the glass slide, smooth side down, onto the fluorimeter.
Step 2- Adjust the height of the phone and fluorimeter so that the picture will be of the side of the drop.
Step 3- Using the micro-pipette, place 80µL of the SYBR GREEN I on the slide, connecting the first two circles in the middle.
Step 4- Again using the micro-pipette, drop 80µL of the sample solution on top of the SYBR GREEN I drop.
Step 5- Position the slide on the fluorimeter, so that the light passes through the center of the drop and focuses on the other side
Step 6- Adjust the phone's distance and focus from the fluorimeter to allow for the best picture.
Step 7- Cover the fluorimeter with the lightbox, leaving one panel open for access.
Step 8- Press the shutter button on the phone and close the lightbox, using a timer on the phone to give you enough time to do so.
Step 9- Once the picture is taken, record which solution is in the photograph.
Step 10- Remove the drop from the slide and dispose of it appropriately.
Step 11- Move the slide to th next position and repeat the previous steps for each sample.


Data Analysis

Representative Images of Samples


Calf Thymus DNA solution concentration of 0 micro grams/mL


With no DNA 2.jpg




Calf Thymus DNA solution concentration of 2 micro grams/mL


With some DNA 2.jpg


Image J Values for All Samples


Calf Thymus DNA Concentration (FINAL), μg/mL ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
0.25 image 1 33353 74.447 2483023 360236
0.25 image 2 33353 85.451 2850044 523085
0.25 image 3 32732 86.562 2833363 707536
0.5 image 1 24263 103.347 2507512 115797
0.5 image 2 24263 105.168 2551685 160892
0.5 image 3 23734 108.302 2570430 179709
1 image 1 20188 157.073 3170995 109397
1 image 2 21164 155.379 3288445 146047
1 image 3 20967 153.903 3226875 151989
2 image 1 26476 188.683 4995584 113277
2 image 2 25922 189.561 4913801 167964
2 image 3 25796 193.791 4999027 142127
5 image 1 27488 204.359 5617421 118420
5 image 2 26257 182.257 4785510 118784
5 image 3 28646 211.319 6053453 226367
0 image 1 38368 90.823 3484683 441468
0 image 2 34253 77.217 2644917 308348
0 image 3 25583 75.811 1939470 514997


Fitting a Straight Line

Flourecence data.jpg