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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OUR TEAM

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

ImageJ negative.jpg

[A sample with DNA (positive signal)]

ImageJ positive.jpg

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
5 10405213 2813 10402400
2 9621172 10633 9610539
1 3329855 27440 3302415
0.5 992867 4311 988556
0.25 1176715 17555 1159160
0 2203865 3767 2200098
Negative1 (0.5) 660226 4104 656122
Negative2 (0.5) 3572442 21428 3551014
Negative3 (0.5) 1198328 2781 1195547
Positive1 (0.5) 5181375 17736 5163639
Positive2 (0.5) 4542836 8532 4534304
Positive3 (0.5) 4847254 6815 4840439
A1 (0.5) 643172 4177 638995
A2 (0.5) 742985 7540 735445
A3 (0.5) 568579 3477 565102
B1 (0.5) 647707 3540 644167
B2 (0.5) 485750 2628 483122
B3 (0.5) 422343 6012 416331
C1 (0.5) 1123599 4395 1119204
C2 (0.5) 2561727 4104 2557623
C3 (0.5) 719651 17872 701779
X1 (0.5) 4696847 5220 4691627
X2 (0.5) 8815318 6399 8808919
X3 (0.5) 8791792 17908 8773884
Y1 (0.5) 7017068 13448 7003620
Y2 (0.5) 4720939 8402 4712537
Y3 (0.5) 9193710 4547 9189163
Z1 (0.5) 6168229 7129 6161100
Z2 (0.5) 5532864 7209 5525655
Z3 (0.5) 5683683 7794 5675889


Fitting a Straight Line

BMEPCRGraph.JPG