BME100 s2014:W Group13 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: Nicole Phillips
Role: Leader, Experimental Protocol Professional
Name: Francis Taguinod
Role:Research and Development Adept
Name: Christian "Chin-Chin" Kaiser
Role: PCR Machine Specialist
Name: Augusta Dumanski
Role: Team Mascot, Experimental Technician
Name: Christiana Montoi-Reeves
Role: Research and Development Guru


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
The SYBR green dye is a dye which glows green when exposed to DNA, specifically the nucleic acids in DNA. The more DNA present, the brighter the glow.


Single-Drop Fluorimeter
The Single-Drop Fluorimeter is a small box with a slot for a multi-welled slide to fit in at the top. There is a blue LED light which shines across where the slide fits in which can be turned on by a switch on the right hand side of the box. The purpose of this flourimeter is to illuminate drops placed on the slide by aligning them properly with the light.


How the Fluorescence Technique Works
The DNA and dye are placed on the slide using a micropipetter in the center of two dots in the middle of the slide. The rough texture of the slide and the holes placed throughout allow the liquid to hold its shape. The slide is then positioned so that the light shines through the drop and a picture is taken in the dark of the drop to get a reading on how much DNA is present based on how bright the sample glows.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: Off
    • ISO setting: normal
    • White Balance: normal
    • Exposure: 200%
    • Saturation: normal
    • Contrast: normal



Calibration

Place the camera a set distance in front of the flourimeter sideways, use the placeholder for the camera to stay in place. Place the box on top of the flourimeter and the phone, set a timer for three seconds and close all flaps. Wait for the camera to snap a picture and remove the box and repeat the steps.


  • Distance between the smart phone cradle and drop = 15 cm


Solutions Used for Calibration [Instructions: See worksheet page 6.]

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Final DNA concentration in SYBR Green I solution (µg/mL)
Dye solution (µL)
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. Set a slide on the fluorimeter for the blue light to be in between two and three holes
  2. Apply a tip on the micropipette
  3. Insert the sample onto the tip
  4. Eject the sample on the slide
  5. Remove the tip and dispose in the proper waste disposal
  6. Take a sample of SYBR Green I on a new tip
  7. Eject the SYBR Green I on the slide with the sample
  8. Properly dispose of the tip after emptying the SYBR Green I
  9. Repeat steps as neccessary


Data Analysis

Representative Images of Samples


Description of image


Sample with DNA:

Description of image



Image J Values for All Samples


Patient Final DNA concentration RawIntDen of Drop RawIntDen of Background drop - background
+ 2.5 2546779 75999 2470780
+ 2.5 2480418 92368 2388050
+ 2.5 2722726 130181 2592545
- 0 523138 98255 424883
- 0 1118946 127085 991861
- 0 679694 51124 628570
tag11 2.5 1007130 96557 910573
tag11 2.5 862403 69095 793308
tag11 2.5 756651 126684 629967
tag12 1 2045247 98443 1946804
tag12 1 4314971 106513 4208458
tag12 1 3499662 132705 3366957
tag13 0.5 3967110 103588 3863522
tag13 0.5 2771929 156282 2615647
tag13 0.5 3536144 135942 3400202
tag21 0.25 3550775 98025 3452750
tag21 0.25 2863309 74523 2788786
tag21 0.25 3753307 70435 3682872
tag22 0.125 3198570 60608 3137962
tag22 0.125 2205818 94114 2111704
tag22 0.125 720263 68615 651648
tag23 0 1035161 87708 947453
tag23 0 1852123 84990 1767133
tag23 0 975350 85293 890057

TABLE GOES HERE


Fitting a Straight Line

Description of image

Our positive control was 4.38 μg/mL

Our negative control was 4.94 μg/mL

Patient 88350 did not show any sign of DNA screening, as the values did not add up to the positive control.

Patient 65583 did not show any sign of flourescent thereby showing that the patient also did not add up to the positive control.