BME100 s2014:W Group13 L5

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Contents

OUR TEAM

Name: Nicole PhillipsRole: Leader, Experimental Protocol Professional
Name: Nicole Phillips
Role: Leader, Experimental Protocol Professional
Name: Francis TaguinodRole:Research and Development Adept
Name: Francis Taguinod
Role:Research and Development Adept
Name: Christian "Chin-Chin" KaiserRole: PCR Machine Specialist
Name: Christian "Chin-Chin" Kaiser
Role: PCR Machine Specialist
Name: Augusta DumanskiRole: Team Mascot, Experimental Technician
Name: Augusta Dumanski
Role: Team Mascot, Experimental Technician
Name: Christiana Montoi-ReevesRole: Research and Development Guru
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)
580802.5
280801
180800.5
0.580800.25
0.2580800.125
080800

[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
+2.52546779759992470780
+2.52480418923682388050
+2.527227261301812592545
-052313898255424883
-01118946127085991861
-067969451124628570
tag112.5100713096557910573
tag112.586240369095793308
tag112.5756651126684629967
tag1212045247984431946804
tag12143149711065134208458
tag12134996621327053366957
tag130.539671101035883863522
tag130.527719291562822615647
tag130.535361441359423400202
tag210.253550775980253452750
tag210.252863309745232788786
tag210.253753307704353682872
tag220.1253198570606083137962
tag220.1252205818941142111704
tag220.12572026368615651648
tag230103516187708947453
tag2301852123849901767133
tag23097535085293890057

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.




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