BME100 f2013:W900 Group11 L5

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OUR TEAM

Name: Diana Terreros
Name: John Sherman
Name: Alvaro Rascon
Name: Timothy Millea
Name: student
Name: student


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is a dye that is used as a nucleic acid stain in PCR and other biological and biochemistry procedures. The dye binds to DNA, which causes it to absorb the blue light emitted by the fluorimeter and to radiate a green light. The dye can bind to double stranded DNA the best, but can also bind to single stranded DNA and RNA. In this lab, it is used to quantify the DNA that resulted from the PCR.


Single-Drop Fluorimeter
The fluorimeter is a small, black box with a part on top of it where the slide can slide in. It has a light switch to turn on the blue light, which shines through the sample that is put on the slide. There is a dial on the left side helps adjust the light so that it shines through the sample. It also comes with a stand where you can put your phone to take pictures of the sample and a file box to cover it while the picture is being taken.

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How the Fluorescence Technique Works
The technique works by adding the SYBR green dye and seeing how much green light is emitted by the sample. The more green light that is emitted, the higher the concentration of DNA is. The slides that are put into the fluorimeter have a particular surface that allows the drops of sample to remain spherical. This allows the blue light to shine through the sample and emit the green light.



Procedure

Smart Phone Camera Settings
We used an Apple iPhone 4 to capture the images.

  • Flash: Off
  • ISO setting: NA
  • White Balance: NA
  • Exposure: NA
  • Saturation: NA
  • Contrast: NA


Calibration

To set up the camera in front of the fluorimeter, we placed it close enough so that it could focus on the sample. We had to angle it right in order to catch the green light in the picture. To do this, we used plastic plates to raise the fluorimeter so that the drop was aligned with the camera. We placed bubble wrap in front of the camera to keep it from sliding down the camera tray. We also used a black file box around the fluorimeter while taking the picture so that the light would show up better in darkness.

  • Distance between the smart phone cradle and drop = 4 centimeters

Bme3.jpg

Solutions Used for Calibration [Instructions: See worksheet page 6.]

0ug/mL DNA 0.125ug/mL DNA O.25ug/mL DNA 0.5ug/mL DNA 1ug/mL DNA 2.5ug/mL DNA
0ug/mL DNA 0.125ug/mL DNA O.25ug/mL DNA 0.5ug/mL DNA 1ug/mL DNA 2.5ug/mL DNA
0ug/mL DNA 0.125ug/mL DNA O.25ug/mL DNA 0.5ug/mL DNA 1ug/mL DNA 2.5ug/mL DNA

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Placing Samples onto the Fluorimeter 1: Place the slide in the fluorimeter. 2: Place 80 uL of the SYBR Green Dye in the middle of the two dots in the center of the rough superhyrdrophobic side of the slide. 3: Place 80 uL of the given calibration DNA concentration sample on the same bead."' 4: Set the timer of the camera and place the shroud over the fluorimeter before the picture is taken.


Data Analysis

Representative Images of Samples

NoDNA.jpg [No DNA]

DNAjj.jpg [Image with DNA]


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
2.5 image 1 12132 155.648 1888318 25782 1862536
2.5 image 2 12440 143.801 1788887 27628 1761259
2.5 image 3 11826 132.418 1565973 24397 1541576
1 image 1 8591 107.693 925190 17818 907372
1 image 2 19157 93.208 1785595 48870 1736725
1 image 3 17240 94.223 1624412 37115 1587297
0.5 image 1 13538 63.055 853636 25964 827672
0.5 image 2 10262 59.546 611057 21179 589878
0.5 image 3 10826 60.995 660334 19538 640796
0.25 image 1 13056 31.483 411043 26221 384822
0.25 image 2 13564 24.411 331109 23243 307866
0.25 image 3 15824 33.638 532288 32853 499435
0.125 image 1 13056 2.146 29046 27009 2037
0.125 image 2 13056 2.146 28023 26674 1349
0.125 image 3 12440 3.857 47980 26855 21125
0 image 1 12440 2.393 29763 25130 4633
0 image 2 11404 2.134 24340 21879 2461
0 image 3 7170 2.864 20532 14359 6173

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