BME100 s2014:T Group8 L5

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Contents

OUR TEAM

Name: Nicole Plachecki
Name: Nicole Plachecki
Name: Omar Benitez
Name: Omar Benitez
Name: Colton Tucker
Name: Colton Tucker
Name: Hooyoung Kim
Name: Hooyoung Kim
Name: Callaway Freeland
Name: Callaway Freeland


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Dye is a photosensitive molecular dye that exhibits a green fluorescence in the dark when added to enough single-stranded DNA.


Single-Drop Fluorimeter
A Single Drop Fluorimeter is a device that shines blue light through a drop sample that is placed on a glass slide and detects fluorescence.

Single Drop Fluorimeter.
Single Drop Fluorimeter.


How the Fluorescence Technique Works
Since using the naked eye to view if a sample has DNA is near to impossible; a fluorescent dye is used. A camera makes the florescent dye even easier to see, and creates images which can be analyzed for amount of dye present.

The way the fluorescence works is by utilizing a green dye that activates only under the presence of double stranded DNA (or dsDNA).



Procedure

Smart Phone Camera Settings
[Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).]

    • Type of Smartphone: Samsung Galaxy S2
    • Flash: Off
    • ISO setting: Default
    • White Balance: Auto
    • Exposure: Highest
    • Saturation: Default
    • Contrast: Default


Calibration

The camera phone was set up in the cradle with an angle to view the side of the drop. The fluorimeter was raised about 1 1/4 inches to accomodate for the phone height.

  • Distance between the smart phone cradle and drop = 6.5 cm

The phone was removed from this image to simplify and completely show distance setup.

Distance from Fluorimeter to Cradle.
Distance from Fluorimeter to Cradle.


Solutions Used for Calibration

Distance of Phone from Fluorimeter 6.5 cm
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR Green I Dye Solution Final DNA concentration in SYBR Green I Solution (μg/mL)
5.0 80 80 2.5
2.0 80 80 1.0
1.0 80 80 0.5
0.5 80 80 0.25
0.25 80 80 .0125
0.0 80 80 0.0


Placing Samples onto the Fluorimeter:

1. Turn on the flourimeter. Place the super hydrophobic slide on the flourimeter. On the hydrophobic side of the flourimeter, place a 80 microliter drop of SYBR GREEN I on the middle rows of the slide.

2. Now on the sphere of dye that is on the slide, add 80 microliter of one of the solutions being tested. This will now react or not.

3. Next the flourimeter will shine a blue light, align the slide so that the drop sample is in the middle of this light.

4. Acquire a box that can cover the flourimeter in order to create a dark environment. Place a smartphone in the cradle, and use a timer on the camera to take a picture of the drop. Once the timer starts quickly put the box over the whole setup, so that outside light does not interfere.




Data Analysis

Representative Images of Samples

High DNA - using green channel Image:drop1.png


Low DNA - using green channel Image:drop2.png

High DNA Concentration Low DNA COncentration
Positive Control Negative Control


Image J Values for All Samples

Trial Final DNA Concentration Area Mean Pixel Value Rawintden of the Drop Rawintden of the Background RAWINTEDEN (drop) - RAWINTDEN (background)
Trial 1 0 91382 71.613 6544125 18757 6525368
0.25 83776 40.875 3424354 2865 3421489
0.5 64632 64.815 4189144 373 4188771
1 72068 74.307 5355152 2461 5352691
2 74956 116.601 8739909 8280 8731629
5 81146 164.265 13329423 5734 13323689
Trial 2 084384 53.044 4476036 2665 4473371
0.25 60532 57.136 3458560 1701 3456859
0.5 88468 42.136 3727677 209 3727468
1 84626 85.175 7208044 106 7207938
2 87906 99.08 8709740 174 8709566
5 89884 162.199 14579066 87 14578979
Trial 3 0 97260 45.051 4381686 7168 4374518
0.25 92784 54.433 5050546 593 5049953
0.5 88652 69.382 6150810 176 6150634
1 111748 73.74 8240277 783 8239494
2 85960 100.872 8670951 175 8670776
5 93351 168.424 15722556 194 15722362

PCR Results with Corrected COncentration Values

PCR Tube Average INTDENS Value PCR Product Concentration (µL/mL) Corrected PCR Product Concentration (µL/mL)
P1A2744551-0.127724333-1.532692
P1B2653457-0.173271667-2.07926
P1C2690172-0.154914-1.858968
+C+ 181239807.56199016790.743882
P2A2786881-0.106559667-1.278716
P2B32088320.1044161.252992
P2C1965640-0.51718-6.20616
-C- 2538375-0.230812667-2.769752


Fitting a Straight Line

Intends values for each Calf Thymus DNA concentration.

Intends values for each Calf Thymus DNA cocnentration.

PCR Results Summary

A Polymerase Chain Reaction (PCR) was completed for 8 DNA samples--3 DNA samples from each of two patients and a positive and a negative control sample. After the reactions proceeded and cooled down, the samples were tested on a fluorimeter for amplification of DNA using SYBR Green I as a stain. The controls were necessary to ensure amplification results for the patients' samples had a comparative standard and the results were due to the procedure and not an external affect. Using a known concentration of DNA from the calibration phase, a standard curve was created that was a basis for conversion from imageJ INTEN values to DNA concentrations for the PCR samples.


Your positive control PCR result was 90.744 μg/mL
Your negative control PCR result was -2.770 μg/mL


Patient 1, ID: 51917 samples hardly showed any visibly show green
Patient 2, ID: 17539 all of their samples showed little to no green dye.


Patient 1, ID: 51917 samples were negative, were a bit blue but had little to no green, and would therefore also be considered negative for the diseased DNA. The average DNA concentration for Patient 1 was -1.824 μg/mL.
Patient 2, ID: 17539 samples were mostly negative, with an average concentration of -2.077, but one of the samples showed a bit of green with a calculated DNA concentration much farther from the negative control than the other results, but still farther from the positive control. The test would be considered negative for the diseased DNA, but with the one sample in disagreement with the other two, a retest may be desired to ensure that the patient is negative for the diseased DNA.



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