BME100 f2013:W1200 Group6 L5

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OUR TEAM

Jenny Chen
Tracy Lopez
Nayobe Bivins
Alex Bugarin
Nicholas Kilpatrick


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
The SYBR green dye is an important component of the fluorescence technique for detecting the amplified target DNA during a PCR (polymerase chain reaction) experiment. An asymmetrical cyanine dye, SYBR green dye is used as a nucleic acid stain because it detects the presence of dsDNA (double-stranded DNA) and only binds to its structure. After being bound to dsDNA it will absorb blue light and emit a bright green color. In contrast, the SYBR green dye produces a weak fluorescence when other molecules such as water or single-stranded DNA are present. The dye can also produce a weak fluorescence when there is RNA present. Therefore, when used with devices like the Single-Drop Fluorimeter, the SYBR green dye can show the presence of DNA in a solution. It can also be used in gel electrophoresis or used to stain agarose gels in order to help visualize the DNA better.

Single-Drop Fluorimeter

Fluorimeter6pm.JPG

The fluorimeter device is a laboratory instrument that serves as a DNA or protein quantification device. The fluorimeter device accurately measures the amount of fluorescence present in a sample. The fluorimeter can measure the intensity and wavelength of the emitted light. It is widely used in applications such as PCR.

The "drop" Fluorimeter device used in this lab was a black box with a space in the middle. The space in the middle was used to easily slide in the hydrophobic glass slides. The hydrophobic glass slide was multi-welled to help with placement of our drop. The slide is coated in Teflon which allows the sample to maintain its shape in a spherical drops on the surface of the slide. The Hydrophobic slides were used so that the drops of DNA could stay as a bubble. It is able to stay as a bubble due to the hydrophobic properties. There is a switch on the right side of the box and when it is flipped, the fluorimeter transmits a blue LED light. The glass slide is positioned so that the blue light goes through the DNA sample.

How the Fluorescence Technique Works
The technique used in the lab detected the fluorescence of the light using wavelengths. The way the fluorometer works is by first mixing the DNA and with the SYBR Green dye. The mixed substance is placed onto the middle of the hydrophobic glass slide, which is placed in the fluorometer. The switch is turned on so that the blue LED light appears. The slide is positioned so that the blue light goes through the DNA sample on the slide. The blue LED light interacts with the green dye which emits a green color. Now the DNA will light up with a certain shade of green and that depends on the concentration of the DNA that was placed on the glass slide. The darker or brighter the green color is indicates a higher concentration of DNA. With images taken of the fluorescence DNA, it can be calibrated and analyzed to see the results of how intense the fluorescence is. Which can be used to see about how much the DNA concentration was in the substance.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S4
    • Flash: None
    • ISO setting: 800 ISO
    • White Balance: Auto
    • Exposure: 2.0
    • Timer setting: 5.0 seconds
  • No additional Smartphone was used


Calibration
The flourimeter was raised using a white plastic box. The camera was placed in the cradle in front of the fluorimeter and the camera application selected. There was a black box covering the whole set to not have any outside source of light. Based on the view of the blue light, the camera position was adjusted until the droplet would be large and centered on the screen. The camera phone was set to focus on the drop. The distance between the drop and cradle was then measured.


PhoneFluoSetup26pm.JPG

A timer setting was used to create as light-secure setting as possible. Three photos were taken and analyzed wit ImageJ.


  • Distance between the smart phone cradle and drop: 9.7 cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (µg/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 (µg/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. Place the slide smooth side down in the fluorimeter
  2. Set the micropipet to 80 microliters and put on a new tip.
  3. Put 80 microliters of SYBR GREEN I solution in between the first two rows of the slide, with the blue light passing through the droplet.
  4. Using a new tip, put 80 microliters of one of the calf thymus dilution onto the same droplet.
  5. Focus the camera phone on the drop and set the camera timer.
  6. Cover the fluorimeter with the black box.
  7. Take three pictures of each drop to be analyzed in imageJ software.
  8. Use the micropipet set at 160 microliters to remove each sample from the slide.
  9. Repeat this process with all the designated calf thymus solutions.


Data Analysis

Representative Images of Samples

Image of drop with no DNA: negative signal
Conc0green.jpg

Image of drop with DNA: positive signal
Conc5green.jpg


Image J Values for All Samples


Calf Thymus DNA Concentration (FINAL), ìg/mL Image AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN - BACKGROUND
2.5 Group6Conc5.jpg 155640 156.535 24363157 129814 24233343
2.5 Group6Conc5Pic2.jpg 146804 144.342 21189975 52893 21137082
2.5 Group6Conc5Pic3.jpg 139396 143.53 20007563 98923 19908640
1 Group6Conc2Pic1.jpg 168304 105.241 17712539 154616 17557923
1 Group6Conc2Pic2.jpg 160336 108.046 17323630 13078 17310552
1 Group6Conc2Pic3.jpg 166560 108.104 18005867 51001 17954866
0.5 Group6Conc1Pic1.jpg 154902 63.077 9770677 53939 9716738
0.5 Group6Conc1Pic2.jpg 144268 65.413 9437067 86282 9350785
0.5 Group6Conc1Pic3.jpg 157834 64.486 10178120 54290 10123830
0.25 Group6Conc0.5Pic1.jpg 157236 46.207 7265369 58401 7206968
0.25 Group6Conc0.5Pic2.jpg 174990 46.154 8076559 85170 7991389
0.25 Group6Conc0.5Pic3.jpg 153700 46.804 7193718 54061 7139657
0.125 Group6Conc0.25Pic1.jpg 176180 21.629 3810623 87020 3723603
0.125 Group6Conc0.25Pic2.jpg 171193 19.744 3379991 34984 3345007
0.125 Group6Conc0.25Pic3.jpg 175499 17.743 3113945 128292 2985653
0 Group6Conc0Pic1.jpg 157512 45.899 7229573 61120 7168453
0 Group6Conc0Pic1.jpg 140430 52.219 7333154 45801 7287353
0 Group6Conc0Pic1.jpg 150408 45.277 6809958 41408 6768550


Fitting a Straight Line

DNAtrend1.JPG