BME100 s2014:W Group14 L5

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Contents

OUR TEAM

Name: Jose Duran
Name: Jose Duran
Name: Shawn Garcia
Name: Shawn Garcia
Name: Christian Keefer
Name: Christian Keefer
Name: Karthik Puncha
Name: Karthik Puncha
Name: Austin Tielke
Name: Austin Tielke

LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green dye was the indicator that was utilized in the experiment in order to detect particular strands of DNA. When added to a substance, we can determine the presence of double-stranded DNA. This type of florescent dye does not necessarily turn the substance green, instead it emits a green light when the appropriate wavelength of light shines on it. The reasoning behind this phenomenon is because the florescent dye strongly binds to double stranded DNA, making it easier to visualize it and detect it. Although it can easily detect the presence of double-stranded DNA, the SYBR Green Dye fluoresces very weakly or may not even fluoresce at all in substances such as water, or in the presence of single-stranded DNA.


Single-Drop Fluorimeter
The Single-Drop fluorometer was the instrument that was utilized in the process of our experiment. This device is generally used to measure the parameters of fluorescence. The device is composed of a portable light box which is used to shine light through the SYBR Green solution, and a black portable box that makes it easy to capture pictures in the dark. In this experiment, our personalized fluoromoter was used to detect the emission of SYBR Green when added to a specific substance. If double-stranded DNA was detected, the light shining through the substance would turn green.


How the Fluorescence Technique Works
In the experiment, we used a single-drop fluorometer to detect the presence of double stranded DNA. This process is composed of a small slide that has a superhydrophobic surface (surface that repels water), and a light box used to visualize the type of light reflected from the drop. When high concentrations of DNA were added to the SYBR Green solution, all wavelengths of light were absorbed except green. However when single-stranded DNA or water was added, the blue LED-light did not illuminate any particular color.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone 5S
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: High
    • Saturation: High
    • Contrast: Low

Calibration

The camera was set up by placing the iPhone 5S in the holding compartment of the lab equipment. It was measured to be 8 centimeters away from the origin of the light emission. The settings (stated above) were adjusted; the height of the fluorimeter was adjusted in order to achieve a perfect side-angle perspective of the "beach ball" shaped liquid drop.

  • Distance between the smart phone cradle and drop = 8 centimeters


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (μg/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Final DNA concentration in SYBR Green I solution (μg/mL)
5 80 80 2.5
2 80 80 1.0
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. Before placing samples onto the fluorimeter, turn on the Blue LED light & have the camera on the smartphone ready.
  2. Insert the glass sampling surface, with the smooth side facing down.
  3. Position the glass sampling surface so that the light emits between the first two rows of the glass.
  4. Place an 80 μL drop of SYBR Green I in the middle of the first two rows.
  5. Swap out the pipette tip with a new tip.
  6. Place an 80 μL drop of a specifically concentrated Calf Thymus DNA solution onto the top of the previously placed SYBR Green I drop. Every three 160 μL sample drops will vary in concentrated Calf Thymus DNA solution.
  7. Place the black screen over the camera, set the camera to take a picture (on a timer) after the lid is shut, take the picture, and proceed towards the next sample drop.
  8. In order to place a new sample drop, remove the 160 μL solution, move the glass sampling surface to the next two available rows, and repeat the process of placing two new 80 μL drops onto each other (as stated before).


Data Analysis

Representative Images of Samples

Sample w/o DNA




Sample w/ DNA



Image J Values for All Samples


Final DNA Concentration in SYBR Green I Solution (μg/mL) Area (pixels) Mean Pixel Value RAWINTDEN of the Drop RAWINTDEN of the Background RAWINTDEN Drop - RAWINTDEN Background
0.56045285.15144216131871514234462
0.55987184.12445215611754124346149
0.56024182.15341598781798543980024
1.063157120.45159043021954545708848
1.064120124.02160482481879455860303
1.063785117.87962484511984546049997
2.573482164.54685588102124168346394
2.574125170.21588215412054518616090
2.573956177.15482584952154118043084
0.0562357.54632015621678423033720
0.0563455.24131584721549523003520
0.0571206.54235849561512313433725
0.1255732522.1451124652165451959201
0.1255769424.56414135861678511245735
0.1255712027.15612484121702451078167
0.255850378.15139521521686723783480
0.255987184.456137545461715413583005
0.255921280.54141321421721643959978

Fitting a Straight Line


PCR Results Summary

TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS " " AVERAGE INTDENS VALUE
Dye solution (µL)
A00 80 80 17514607.24 16358382.85 13105657.98 15659549.36
A01 80 80 7527051.87 5439907.836 5817272.88 6261410.862
A02 80 80 5780187 5264154.775 7535894.664 6193412.146
A03 80 80 7493409.77 6729272.54 6821746.185 7014809.498
B00 80 80 1885223.456 1780211.712 1991211.874 1885211.816
B01 80 80 6901246.104 8036257.608 9159860.332 8032454.681
B02 80 80 8841431.624 10061646.59 9186306.417 9363128.21
B03 80 80 10232940.21 11665080.57 10564810.97 10820943.92



PCR Product Tuble Label Average INTDENS Value PCR Product Concentration (μg/mL) Corrected PCR Product Concentration (μg/mL)
A0015659549.3610.531894650.877657887
A016261410.8622.9527506950.246062558
A026193412.1462.8979130210.241492752
A037014809.4983.560330240.296694187
B001885211.816-0.576442084-0.04803684
B018032454.6814.381011840.36508432
B029363128.215.4541356530.454511304
B0310820943.926.6297934840.55248279





Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.

Your positive control PCR result was 0.877657887 μg/mL
Your negative control PCR result was -0.04803684 μg/mL

Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed.

Patient 26734: average value of 0.261416499 μg/mL
The images of the sample drops had little accents of green that were present.

Patient 22707: average value of 0.3457359472 μg/mL
The images of the sample drops had a small hint of green, yet still greater than Patient 26734.

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.

Patient 26734: The results concluded that the test was inconclusive because Patient 26734 produced results that had a substantial amount of PCR Product Concentration; however, not great enough to conclude that the patient contains the sought-out after DNA strands.
Patient 22707: The results concluded that the test was inconclusive because Patient 22707 produced results that had a greater PCR Product Concentration than Patient 26734; however, not great enough to conclude that the patient contains the sought-out after DNA strands.


Each patient was found within the middle of the range between the positive control (0.877657887 μg/mL) and the negative control (-0.04803684 μg/mL); thus, further tests would need to be performed in order to reach a conclusion on whether or not these two patients may or may not have the DNA strands that the lab investigation was intended towards looking for.

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