BME100 s2014:W Group3 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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Contents

OUR TEAM

Name: Joseph Roskop
Name: Joseph Roskop
Name: Lorelei Fyfe
Name: Lorelei Fyfe
Name: Ana Kenia Viera
Name: Ana Kenia Viera
Name: Ben Lenschow
Name: Ben Lenschow
Name: Adnan Alsharif
Name: Adnan Alsharif
Name: Group Picture
Name: Group Picture


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green Dye is a nucleic acid stain used to identify isolated segments of DNA. Usually utilized to determine whether or not genetic pathogens or discrepancy's are present. Capable of detecting Virus's like HIV as well as precursors for Diabetes.

Single-Drop Fluorimeter
The Single-Drop Fluorimeter is the device that illuminates the dye allowing for both qualitative and quantitative analysis of the compounds being observed. The device itself consists of just a box stand with a slide holder on its surface. Lateral to the stand is a small fluorescent bulb that illuminates the fluid solution. On the side of the box is a flip switch that turns the fluorescent light on and off.


How the Fluorescence Technique Works
The Fluorescence Technique's function is to identify the presence of different genetic pathogens. This works by bathing the SYBR Green Dye and DNA sample with a black light. A phenotype present in target DNA sequences causes positively effected DNA samples to glow with a green fluorescence when subjected to the black light.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S4
    • Flash: Inactive
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: Highest Setting
    • Contrast: Lowest Setting


Calibration


The smart-phone is set in a position so that its camera is eye level with the slide. In order to achieve this with phones of different lengths, it is necessary to raise the box off the ground using small booklets or pencil boxes. For the most effective images the phone timer is set so that the image is taken after the lid of the blackout box is closed.

  • Distance between the smart phone cradle and drop = 14cm


Solutions Used for Calibration

Initial Concentration (micrograms) Volume of the 2x DNA solution Volume of SYBR GREEN 1 FInal DNA concentration in SYBR GREEN 1 solution
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

[Add more rows as needed]


Placing Samples onto the Fluorimeter

  1. Place a slide with the smooth side back down in the cut out slot in the flourimeter.
  2. Place the cellphone on the cradle with the camera facing the flourimeter.
  3. Adjust the flourimeter to the necessary height using booklets or trays.
  4. Measure the distance between the cellphone and the flourimeter.
  5. Put a 80 microliter drop of SYBR Green 1 on the slide on the flourimeter directly across from the fluorescent light bulb.
  6. Add another 80 microliter drop of your sample on the top of the drop already on the slide.
  7. Make sure that the light is switched "ON"
  8. Using the cellphone and timer press the capture button on the phone.
  9. Put down the lid of the light box.
  10. Allow phone to take the picture and then lift the lid.
  11. Record what sample you just took a picture of and repeat this with the next position.


Data Analysis

Representative Images of Samples

[A sample with no DNA]

[A sample with DNA (positive signal)]

PCR Results Summary


Eight PCR reactions were completed in previous lab. This lab used the SYBR Green I and imaging technique to clearly see the amount of amplified DNA in each reaction. A standard curve converted the INTDEN values to DNA concentration. The threshhold values were measured by the controls of the positive and negative PCR reactions to determine if the two patients in this experiment were positive or negative.
Positive control : PCR result 2,428,578 μg/mL
Negative control : PCR result 935,166 μg/mL
Patient 80926 : A basic clear shiny drop : Recordings were 325,798 μg/mL, 259,300 μg/mL, and 734,163μg/mL. These numbers are smaller compared to the positive control value and closer in comparison to the negative control. This concludes that the first patient had a negative test result.
Patient 83588 : A bright green fluid drop : Recordings were 3,717,326 μg/mL, 3,488,620 μg/mL, and 2,897,463 μg/mL. These numbers are larger in comparison to the negative control value and closer in comparison to the positive control value. This concludes that the second patient had a positive test result.

Image J Values for All Samples


Final DNA Concentration in SYBR Green I Solution (μg/mL) RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDENdrop - RAWINTDENbackground
510405213281310402400
29621172106339610539
13329855274403302415
0.59928674311988556
0.251176715175551159160
0220386537672200098
Negative1 (0.5)6602264104656122
Negative2 (0.5)3572442214283551014
Negative3 (0.5)119832827811195547
Positive1 (0.5)5181375177365163639
Positive2 (0.5)454283685324534304
Positive3 (0.5)484725468154840439
A1 (0.5)6431724177638995
A2 (0.5)7429857540735445
A3 (0.5)5685793477565102
B1 (0.5)6477073540644167
B2 (0.5)4857502628483122
B3 (0.5)4223436012416331
C1 (0.5)112359943951119204
C2 (0.5)256172741042557623
C3 (0.5)71965117872701779
X1 (0.5)469684752204691627
X2 (0.5)881531863998808919
X3 (0.5)8791792179088773884
Y1 (0.5)7017068134487003620
Y2 (0.5)472093984024712537
Y3 (0.5)919371045479189163
Z1 (0.5)616822971296161100
Z2 (0.5)553286472095525655
Z3 (0.5)568368377945675889


Fitting a Straight Line



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