BME100 f2013:W1200 Group5 L5

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Contents

OUR TEAM

Name: Dominick Cocciola
Name: Dominick Cocciola
Name: Hany Arafa
Name: Hany Arafa
Name: Wendy Gray
Name: Wendy Gray
Name: Kristina Roscher
Name: Kristina Roscher
Name: Estefania Meza
Name: Estefania Meza

LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green dye is a small molecular dye that fluoresces in the presence of DNA but fluoresces weakly in the presence of water or single strands of DNA. The green dye detects double-stranded DNA generated during PCR. It is also used to detect DNA in gel electrophoresis.


Single-Drop Fluorimeter

Image:fl1.jpg
The Single-Drop Fluorimeter is a device used to measure fluorescence. The base of the fluorimeter houses a slide holder specialized to hold a rectangular glass slide that has a hydrophobic coating. This coating allows water to collect in a droplet on the surface that will be used to measure the fluorimeter's light source, a blue LED light. The base also allows a camera to sit in front of the slide. Finally, a light box is placed over the base that encloses the fluorimeter and prevents light from outside sources from entering, making the droplet and LED more visible for the camera to capture.


How the Fluorescence Technique Works
Fluorescence is a process in which the emission of light from a sample droplet is measured in order to collect data. For this experiment specifically, a blue LED is passed through a droplet containing a sample of DNA and SYBR Green dye which will fluoresce when interacted together. This LED light excites the molecules within the droplet causing the droplet to emit light in a more focused wavelength which can be measured visibly when captured in a picture using a camera. On a more theoretical level, the light given off by the droplet is measured by the equation of fluorescence, which states that the product of the intensity of the LED, the absorption coefficient and molar concentration of SYBR Green dye, and a value of quantum efficiency, equals the fluorescent intensity. This fluorescent intensity is then related to the concentration of DNA in the droplet when removing the background fluorescence of non-reacting molecules (Week10 Lab Handout-BME 100).



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5s
    • Flash: off
    • ISO setting: n/a
    • White Balance: n/a
    • Exposure: n/a
    • Saturation: n/a
    • Contrast: n/a


Calibration


Image:fl2.jpg

  • Distance between the smart phone cradle and drop = 5 cm


Solutions Used for Calibration

Calf Thymus DNA (μg/mL) SYBR Green Dye Volume (μL)2X DNA Solution Volume (μL) Final DNA Concentration (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. Step One: Insert a glass slide into the fluorimeter with the "smooth side" facing down.
  2. Step Two: Extract X ml of SYBR green fluid and place it on the sample slide between the First and Second row on the middle dots.
  3. Step Three: Extract and place X ml of the sample concentration that is to be tested on the same fluid that was used int he previous step.
  4. Step Four: After each photo has been taken, remove the sample fluid and dispose of it as instructed.
  5. Step Five: Move the slide into position in such that the blue light is directed onto the middle of the next 2 dots that have not been used.
  6. Step Six: Repeat steps 1-5 until all samples are tested. Remember to use a new slide when each dot has been used on the slide.


Data Analysis

Representative Images of Samples

Image:NODNAOVAL2.jpg


Image:DNAOVAL.jpg
Positive Signal




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 RAWINTDEN - BACKGROUND
2.5image 15717591.47650175241014474916077
2.5image 25660689.23250510601583294892731
2.5image 35798094.2595465111935885371523
1image 15479292.85150874711073974980074
1image 25800277.4454491953909634400990
1image 35383574.7864026096929743933122
0.5image 15109377.1553942062827233859339
0.5image 24982283.8884179470758904103580
0.5image 35241286.0554510312904074419905
0.25image 14903685.1964177657806634096994
0.25image 25492688.9034883089959124787177
0.25image 34994078.763933264931433840121
0.125image 14686154.9742576142883212487821
0.125image 25106653.4093238055892183148837
0.125image 35666487.33749488821002754848607
0image 14703246.1632171138767742094364
0image 25182449.352557534773912480143
0image 34697657.16526853741781792507195


Fitting a Straight Line
Image:Plot1.PNG

Disclaimer: It is also worth noting that the RAWINTDEN values are much lower than the theoretical values. In addition, there is less than optimal change of the RAWINTDEN values between the concentrations of the DNA. Even though the difference is minute, it is still measurable, and these data points can still be used for a calibration as long they are consistent with the values in the next experiment using the actual PCR DNA.

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