BME100 s2014:W Group5 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

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Name: Garrett Sciascia
Role: Mad Scientist
Name: Juan Bahena
Role: OpenPCR Technician
Name: Blake Marx
Role: WetWare Correspondent
Name: Danielle Hallaran
Role: Data Collection
Name: Naomi Walliman
Role: Team Manager

LAB 5 WRITE-UP

Background Information

SYBR Green Dye
A fluorescent DNA dye that binds all dsDNA and is measured by the change in fluorescence throughout. The dye is used in the PCR reaction in order to detect double stranded DNA. This allows experimenters to quantify the amount of DNA being produced.


Single-Drop Fluorimeter
An instrumentation that is able to detect fluorescence. The quantity is proportional to the number of molecules being deteced.
Fluorimetergroup5.JPG


How the Fluorescence Technique Works
A technique that detects light in a molecule much similar to a spectrometer. It uses an ultraviolet light and excites the electrons causing the molecules to emit light.



Procedure

Smart Phone Camera Settings

First Phone:

  • Type of Smartphone: iPhone 5s
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: Highest Settings
    • Contrast: Lowest Settings


Additional Phone:

  • Type of Smartphone: Galaxy Samsung S3
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: Highest Setting
    • Contrast: Lowest Setting


Calibration

First turn on the Blue LED switch in order to activate the excitation light. Also make sure the camera is open with the settings listed above. From there place the pone on the cradle with 90 degree angle from the slide. Make sure to adjust the height of phone and/or fluorimeter in order to take pictures of the drops. Lastly, the cradle should be as close to the fluorimeter as possible with having a clear picture still.

  • Distance between the smart phone cradle and drop =

4.5 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Dye solution (μL) Final DNA concentration in SYBR Green I solution (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 0


Placing Samples onto the Fluorimeter

  1. On the first two rows of the slide, place one drop (the pipette is calibrated to 80 microlitres) of SYBR Green.
  2. Add a second drop of the diluted calf thymus DNA on top of the first drop on the slide. We now have a sample!
  3. Adjust the slide such that the blue LED light is using the drop to focus on the other side of the drop.
  4. Use the camera's timer to take a picture after closing the light box (this prevents as much outside light as possible from contaminating our data).
  5. Remove the box, being careful not to knock the smartphone out of position.
  6. Use the pipette to remove the entire 160 microlitre drop from the slide, then move the slide into the next position and use the next concentration standard.
  7. Repeat as needed.


Data Analysis

Representative Images of Samples

Negative Signal
N1.png

Positive Signal
P1.png


Image J Values for All Samples

PCR Product TUBE LABEL Diluted Volume (µL) SYBR GREEN Volume (µL) INTDEN 1 INTDEN2 INTDEN3 AVERAGE INTDENS VALUE
A1 80 80 2716687 3229970 3340085 3095581
A2 80 80 1451878 2568844 8446955 4155892
A3 80 80 3862714 8011744 1600043 4491500
B1 80 80 9856858 5058003 3108259 6007707
B2 80 80 6862644 5685363 6783718 6443908
B3 80 80 6287959 7119245 9598254 7668486
+ 80 80 11779932 7973600 11270538 10341357
- 80 80 4371681 2482949 1977785 2944138
Fitting a Straight Line

Linear Regression of PCR Results
Dna calibration graph.jpg


Interpretation

Based on the results from the image analysis, the intensity in the green coloration in patient B was higher than patient A. Due to the fact that false positives and negatives were controlled for, it can be confidently said that patient B's DNA indicated a positive result for gene detection test. Patient A did not test positive. According to the calibration, it should be noted that the concentration of DNA is positively correlated to the green intensity of the SYBR Green dye; thus the high intensity in patient B's amplified DNA would be expected to increase with increased concentration of DNA.