BME100 f2013:W900 Group10 L5

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OUR TEAM

Name: Joslin Jose
Name: Barrett Anderies
Name: Liam Williams
Name: Duran Charles


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SBYR Green I dye is a cyanine dye used as a nucleic acid stain. When bound to DNA it absorbs blue light and emits green light. The stain binds to double stranded DNA (dsDNA) at very high levels and binds to single stranded DNA at much lower levels. This allows us to measure the amount of double stranded DNA while getting minimal noise on our signal from the presence of single stranded DNA. It can also stain RNA at lower levels, but this is not important for our experiment (in which we know our sample does not contain RNA). It is also the most sensitive stain available for detecting double stranded DNA during PCR.


Single-Drop Fluorimeter

This device is used to excite the stained DNA molecules in order to generate the designed signal, a green light, for us to capture with our camera. The single-drop fluorimeter is designed to hold a single drop of the sample and pass the wavelength ~497nm of light required to excite the stained DNA molecules within the sample.

Bme100 grp10 single.JPG


How the Fluorescence Technique Works

A droplet is placed on the hydrophobic slide which allows the droplet to hold its spherical shape. The single droplet is then exposed to a blue light beam to excite the stained molecules which proceed to emit green light (our signal). We capture this signal (green light) with our smartphone camera. In theory, the amount of signal captured by our camera should be proportional to the concentration of DNA in the sample (the pictures must be filtered so that only the amount of green light captured is taken into consideration). Therefore, once we have calibrated our camera with known concentrations of DNA we should be able to compare signal strengths (green light emittance) from unknown DNA concentration samples with our calibration data to accurately estimate the DNA concentration in that sample.


Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy SIII
    • Flash: OFF
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: +2 (maximum)
    • Saturation: Auto (no manual options)
    • Contrast: Auto (no manual options)

Calibration


The camera was placed upright in the supplied stand. The camera lens was positioned 6cm from the droplet in order to get the closest view possible while still being within the focus range of the camera. The height of the fluorimeter was adjusted so that the lens of the camera was at the same height as the droplet to ensure a full side view.

  • Distance between the smart phone camera lens and drop = 6cm

Bme100 grp10 cal.JPG

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 (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 a clean slide with the hydrophobic side up into the slot in the Fluorimeter.
  2. Turn on the Fluorimeter.
  3. Use a micro-pipette to transfer 80 µL of SYBR Green I Dye and 80 µL of sample (either water or DNA solution) onto the slide to form a single droplet.
  4. Move the slide until the droplet is directly in line with the blue light beam (if not so already).
  5. Position camera at the predetermined position (see "Calibration") and set a countdown timer on the camera.
  6. Focus the camera on the droplet and start the countdown timer.
  7. Cover the entire apparatus with the supplied box to minimize external light and wait for the camera to capture the picture.
  8. Remove the box, open the recently captured picture and rename it to something that represents the sample DNA concentration and picture number.
  9. Remove the 160 µL droplet from the Fluorimeter slide with a micro-pipette and remove any remaining liquid with a paper towel.
  10. Repeat the above steps two more times to get a total of three pictures of three different droplets of the same DNA concentration.
  11. Repeat the above steps for each sample.


Data Analysis

Representative Images of Samples

The picture below shows the control case where no DNA is present in the sample

BME100 Group 10 Drop Without DNA.png

The picture below shows a test case where DNA is present in the sample

BME100 Group 10 Drop With DNA.png


Image J Values for All Samples

CT DNA Final Concentration (µg/ml) Area Mean Pixel Value RAWINTDEN Drop RAWINTDEN Background Corrected INTDEN
0 63850 9.801 625816 59414 566402
0 66618 14.951 996018 79261 916757
0 63749 8.569 546237 63420 482817
0.125 69214 8.492 587772 80722 507050
0.125 67620 21.642 1463410 93467 1369943
0.125 50109 8.395 420689 7013 413676
0.25 63624 30.175 1919850 65755 1854095
0.25 54968 31.24 1717180 58722 1658458
0.25 62254 34.8 2166468 13405 2153063
0.5 64057 76.566 4904612 69973 4834639
0.5 64966 55.922 3633024 15391 3617633
0.5 69714 54.787 3819397 13548 3805849
1 72788 89.233 6495124 62681 6432443
1 71872 89.702 6447038 72261 6374777
1 68364 87.689 5994752 14063 5980689
2.5 74024 124.657 9227622 16445 9211177
2.5 75904 130.099 9875048 84898 9790150
2.5 83280 127.561 10623306 76871 10546435


Fitting a Straight Line

  • The below graphs illustrate the linear correlation between dsDNA concentration and fluorescence. The first graph shows the raw fluorescence data plotted against the known dsDNA concentrations, and the second graph shows the corrected fluorescence data (raw integrated density minus background integrated density) plotted against the known dsDNA concentrations.

BME100 Group 10 dsDNA pre-correction.png

BME100 Group 10 dsDNA post-correction.png