BME100 f2013:W900 Group13 L5

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OUR TEAM

Name: Tyler Angell
Name: Reem Gerais
Name: Andrew Luc
Name: Allison Marley
Name: Cory Riecken


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is a reagent used to stain DNA. After combining the SYBR Green dye with a double stranded DNA (Single stranded DNA works as well, but produces less effective results), the solution will be able to absorb blue light and emit green light. By measuring the green light, one will be able to measure how much DNA is present in the solution.


Single-Drop Fluorimeter

Singledropfluorimeter.jpg
The single-drop fluorimeter device is a box that emits a blue light. A slot is present on the device for a glass slide to be placed on. Droplets of the SYBR Green dye and DNA solutions are placed on the glass slide for the light to be passed through.

How the Fluorescence Technique Works

The fluorescence technique works by combining a fluorescent dye to the DNA. In this lab, SYBR Green dye was used as the fluorescent dye. The droplets will remain in place due to the Teflon coated glass. A beam of blue light is emitted from the single-drop fluorimeter and is passed through the droplet. A fluorescent green light will be emitted from the droplet which will be measured by taking a picture on a smartphone and analyzing the picture file on ImageJ.



Procedure

Smart Phone Camera Settings
Phone: Galaxy S3

  • Flash: Off
  • ISO setting: 800
  • White Balance: Auto
  • Exposure: Maximum
  • Saturation: Maximum
  • Contrast: Minimum

Calibration

Setup.jpg

1. Adjust the camera settings on the smart phone so that they match the settings above.

2. Place smart phone in the cradle.

3. Make sure that the distance from the smart phone and the cradle is 11 cm.

Solutions Used for Calibration

Calf Thymus DNA Concentration (Microg/ml) Volume of the 2X DNA Solution (ul) Volume of the SYBR GREEN I Assay (ng/mL) 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 blank


Placing Samples onto the Fluorimeter

  1. Before the sample can be placed on the Fluorimeter, the fluorimeter must be set up. This is done by first by placing the glass down on the fluorimeter; the glass must be placed so that the rough side is faced up. The fluorimeter must be turned on. Adjust the glass slide so that the blue light shines in the center of two rows. The hieght of the fluorimeter may need to be adjusted so that the camera has a clear view of the light.
  2. For the next steps, the micropipet will be needed. Begin by adjusting the volume on the micropipet to 80μL, and placing a new micropipet tip on the micropipet.
  3. Use the micropipet to gather 80μL of the SYBR Green I to be placed on the slide. The SYBR Green I will be placed on the glass slide for each sample. Be sure that the camera settings are all in place and take photograph of solely SYBR Green I, this is your blank.
  4. Next, use the micropipet to gather 80μL of the first sample, 0.25, and place it into the 80μL SYBR Green I present on the slide. Be sure that the light from the fluorimeter is in line with the droplet.
  5. Photograph the sample and SYBR Green I.
  6. Remove sample from slide with micropipet and repeat steps for each sample. After all samples have been collected (0,0.25,0.5,1,2,5) repeated this process two more times for a total of three pictures per sample.


Data Analysis

Representative Images of Samples


Sample with no DNA
Owwnodnareal.jpg

Sample with DNA
Owwdna.jpg


Image J Values for All Samples

Calf Thymus DNA concentration (Final), Microg/ml AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND Corrected INTDEN
0 23332 25.472 594295 67498 526797
0 22172 30.492 676054 76231 599823
0 24210 15.191 467788 60278 407510
0.125 36784 13.709 542371 49735 492636
0.125 22986 16.12 370541 51296 319245
0.125 21472 10.899 334023 20721 313302
0.25 24824 66.066 1640022 24889 1615133
0.25 23060 74.497 1728268 69004 1659264
0.25 21530 61.485 1323755 35755 1288000
0.5 21572 80.4 2138451 56743 2081708
0.5 26336 76.749 1021273 47896 973377
0.5 25448 95.74 1636374 54779 1581595
1 21796 97.793 2151322 60743 2090579
1 23734 126.199 1995195 74321 1920874
1 27404 119.256 2268082 79832 2188250
2.5 22144 129.478 3512864 89543 3423321
2.5 23589 175.595 4142091 101563 4040528
2.5 26342 131.337 3459687 81664 3378023

Fitting a Straight Line

BeforeCorrection.jpg AfterCorrection.jpg