BME100 f2013:W900 Group7 L5

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OUR TEAM

Name: Roberto A. Aguirre
Name: Christopher G. Cusick
Name: Abby Monhollen
Name: Neel A. Pendyala
Name:Shelby S. Martin

LAB 5 WRITE-UP

Background Information

SYBR Green Dye

The SYBR Green Dye is a small molecular dye that fluoresces, or glows, very well in the presence of dsDNA (double stranded DNA). In this experiment, SYBR Green I was used, which has maximum efficiency/transmittance at a wavelength of approximately 500 nm, which lies in the range for green light. Therefore, when the SYBR Green Dye binds to the dsDNA and is excited, it strongly emits green light. The quantification and measurement of this green light was performed by taking pictures with a smartphone from a constant distance away from the single-drop fluorimeter.

Single-Drop Fluorimeter

Thisdevice.jpg

The single-drop fluorimeter was used to detect the amount of fluorescence in each sample. It is a small, box-shaped device with a thin, long slot. A slide with a rough, superhydrophobic surface and circles of glass was placed in this slot. The device also emits a blue light--the purpose and role of this blue light in the fluorescence technique is explained in the next section.

How the Fluorescence Technique Works

The fluorescence technique is based on the properties of the slides placed in the single-drop fluorimeter device. These slides have a rough surface made of Teflon that is superhydrophobic as well as small circles made of glass. Because DNA is polar, and hence hydrophilic, it is repelled by the superhydrophobic surface and binds to the small glass circles on the slide due to their adhesive properties. Therefore, adding DNA to the slide causes the DNA to form small spherical globules, rather than spread all over the slide. This allows two things to happen: first, light from the Blue LED of the single-fluorimeter device is more strongly focused onto the DNA, thereby exciting the SYBR green dye and increasing the fluorescence event; two, the solvent added along with SYBR Green I forms a complex with DNA and prefers to go to the surface of the globule.


Procedure

Smart Phone Camera Settings

  • Type of Smartphone: HTC EVO 4G LTE
    • Flash: inactive
    • ISO setting: 800
    • White Balance: auto
    • Exposure: highest possible setting
    • Saturation: highest possible setting
    • Contrast: lowest possible setting



Calibration


Theotherthing.jpg

Place the smartphone in the cradle at a right angle from the slide. Adjust the height of the fluorimeter using the plastic trays to achieve the 90° angle. Adjust the distance of the cradle to the fluorimeter so that the phone is as close to the slide as possible so you get a clear picture (minimum of 4 cm). Be careful not to shift the phone and fluorimeter setup too much from its fixed state during the experiment, as it can affect the difference between each pictures of globule samples.

  • Distance between the smartphone cradle and drop = 4.8cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (microg/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR Green 1 Dye solution (μL) Final DNA concentration in SYBR Green 1 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. Place the slide in the single-drop fluorimeter with the superhydrophobic side facing upwards.
  2. Place 80 uL of SYBR Green I solution onto the first two middle circles of the slide using micro-pipette.
  3. Using a new tip, place 80 uL of the sample on top of the SYBR Green I to create a 160 uL mixture.
  4. Adjust the slide so that blue LED light of the fluorimeter passes through the drop and focuses onto the hole on the opposite side.
  5. Once the slide is adjusted, place the box over the device.
  6. When the box is placed on top, take a picture of the sample, making sure a timer of at least 3 seconds is set on the camera.
  7. Wait for the picture to be taken (about 5 seconds) and then remove the box.
  8. With the micro-pipette, remove the sample from the glass slide and dispose of it properly.
  9. Repeat the above steps three times for each sample until all the picture have been taken, using a new placement on the slide for each sample.


Data Analysis

Representative Images of Samples

Drop With No DNA

This image is of a sample containing no DNA.

Drop With DNA

This image is of a sample containing a 2.5 microg/mL solution of DNA.


Image J Values for All Samples

Calf Thymus DNA Concentration (microg/ml) Area ' Mean Pixel Value Rawintden of the Drop Rawintden of the Background
0 88656 23.157 2052977 203071
0 94568 15.307 1447508 403721
0 95932 26.175 2511027 414153
0.125 111212 19.166 2131465 387059
0.125 115664 30.826 3565431 404244
0.125 110392 26.377 2907362 474133
0.25 102036 18.781 1916353 183083
0.25 104172 28.088 2926001 150042
0.25 99520 25.312 2519017 348084
0.5 160720 26.202 4211262 451656
0.5 139792 58.799 8219689 351685
0.5 159932 41.802 6685494 267241
1 119696 121.236 14511451 422557
1 113752 121.352 13804075 269154
1 111020 104.089 11556001 135891
2.5 119588 140.9 16850001 437612
2.5 126096 138.212 17428018 494050
2.5 127696 143.433 18315851 298791


Fitting a Straight Line

GraphLine