Difference between revisions of "BME103 s2013:T900 Group4 L2"

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(Background Information)
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'''How the Fluorescence Technique Works'''<br>
 
'''How the Fluorescence Technique Works'''<br>
The fluorescence technique measures the concentration of DNA in a solution by adding a fluorescent dye and measuring the amount of fluorescence.  This is done using a single drop fluorometer with a Teflon-coated glass slide.  The slide's surface is not completely covered; circles of bare glass remain, which water sticks to, allowing drops of liquids to stay in place.  Drops of DNA solution are placed on the glass circles along with SYBR Green I. Blue LED light from the fluorometer is aimed at the drops, exciting the dye and causing it to fluoresce. This fluorescence is then analyzed to determine the concentration of DNA in the solution.
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The fluorescence technique measures the concentration of DNA in a solution by adding a fluorescent dye and measuring the amount of fluorescence.  This is done using a single drop fluorometer with a Teflon-coated glass slide.  The slide's surface is not completely covered; circles of bare glass remain, which water sticks to, allowing drops of liquid to stay in place.  Drops of DNA solution are placed on the glass circles along with SYBR Green I. Blue LED light from the fluorometer is aimed at the drops, exciting the dye and causing it to fluoresce. This fluorescence is then analyzed to determine the concentration of DNA in the solution.
  
 
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Revision as of 00:38, 2 April 2013

Owwnotebook icon.png BME 103 Spring 2013 Home
People
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Photos
Wiki Editing Help
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OUR TEAM

Name: Kinjal Ahir
Procedure
Name:Zach Young
Initial Machine Testing
Name: Anna Essex
Initial Machine Testing
Name: Tuan Phan
Research & Design
Name: Amelia Lax
Research and Design


LAB 2 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green I is a fluorescent dye that binds to double-stranded DNA and fluoresces green when excited by blue light. SYBR green dye is very safe to users and cheap, making it ideal for basic DNA visualization.

Single-Drop Fluorimeter
A fluorimeter is a device used to measure amounts of fluorescence. Fluorimeters usually measure fixed wavelengths either with filters, monochromators, or fixed diodes. Fluorimeters have two light detectors; one detects absorbance while the other detects fluorescence emission.

Group4 Fluorometer.JPG
Drops of DNA solution on flourimeter slide

How the Fluorescence Technique Works
The fluorescence technique measures the concentration of DNA in a solution by adding a fluorescent dye and measuring the amount of fluorescence. This is done using a single drop fluorometer with a Teflon-coated glass slide. The slide's surface is not completely covered; circles of bare glass remain, which water sticks to, allowing drops of liquid to stay in place. Drops of DNA solution are placed on the glass circles along with SYBR Green I. Blue LED light from the fluorometer is aimed at the drops, exciting the dye and causing it to fluoresce. This fluorescence is then analyzed to determine the concentration of DNA in the solution.


Procedure

Smart Phone Camera Settings

  • Iphone 4
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto


Calibration

  • Place the smartphone on the cradle at a right angle to both the table and the fluorimeter slide
  • Adjust the height of the fluorimeter slide if necessary so the camera is level with the drop
  • Distance between the smartphone cradle and drop = 4cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (μg/mL) Volume of the 2x DNA solution (μL) Volume of the SYBR GREEN I Dye Solution (μL) Final DNA concentration PicoGreen Assay (ng/mL)
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 blank


Placing Samples onto the Fluorimeter

  • Step one: Check/adjust camera settings and turn the flash off
  • Step two: Obtain calf thymus DNA, gloves, lab coat, glass slide, buffer tubes, and SYBR Green I
  • Step three: Slide the glass slide into fluorimeter
  • Step four: Drop 80μL of solution onto slide in front of blue light
  • Step five: Put dark box over fluorimeter and smartphone and take pictures of the drops


Data Analysis

Representative Images of Samples

Negative DNA circle.jpg
Circle analysis of DNA negative drop (0μg/mL)

Positive DNA circle.jpg
Circle Analysis of DNA positive drop (5μg/mL)

As shown, the DNA positive drop is much brighter.


Image J Values for All Samples

Trial DNA Concentration (μg/mL) Area Mean Pixel Value RAWINTDEN (drop) RAWINTDEN (background)
1 0 10,906 21.672 236,351 70,338
1 0.25 10,044 35.971 361,290 42,572
1 0.5 10,562 84.274 890,103 52,974
1 0.5 10,452 50.546 528,303 50,012
1 1 10,350 115.226 1,192,594 39,995
1 2 10,914 193.145 2,107,989 46,923
1 5 10,282 142.563 1,465,554 55,342
2 0 10,808 50.412 544,856 71,106
2 0.25 10,510 51.038 536,412 39,795
2 0.5 10,688 78.799 842,203 57,075
2 1 10,432 90.965 948,944 52,390
2 2 10,103 152.075 1,536,481 61,000
2 5 10,440 115.674 1,207,635 51,873
3 0 10,131 37.264 377,520 66,289
3 0.25 10,448 61.987 647,645 48,887
3 0.5 10,762 64.475 693,875 40,978
3 1 10,465 74.835 783,144 47,549
3 2 10,188 155.63 1,585,554 43,186
3 5 10,672 72.312 771,711 47,142



Fitting a Straight Line
[Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]