BME103 s2013:T900 Group1 L2

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{| style="wikitable" width="700px"
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| [[Image:BME103 Group1 KID.jpeg|100px|thumb|Kristi Norris:<br>Protocol Planner]]
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| [[Image:BME103 Group1 KID.jpeg|100px|thumb|Kristi Norris:<br>Protocol/Procedure]]
| [[Image:BME103_Group1_Carlos2.jpg|100px|thumb|Carlos Renteria:<br>Research and Design Specialist]]
| [[Image:BME103_Group1_Carlos2.jpg|100px|thumb|Carlos Renteria:<br>Research and Design Specialist]]
| [[Image:BME103_group1ram.jpg|100px|thumb|Raul Monzolo:<br>Open PCR Machine Engineer]]
| [[Image:BME103_group1ram.jpg|100px|thumb|Raul Monzolo:<br>Open PCR Machine Engineer]]

Revision as of 00:13, 2 April 2013

BME 103 Spring 2013 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
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Image:BME494_Asu_logo.png


Contents

OUR TEAM

Kristi Norris:Protocol/Procedure
Kristi Norris:
Protocol/Procedure
Carlos Renteria:Research and Design Specialist
Carlos Renteria:
Research and Design Specialist
Raul Monzolo:Open PCR Machine Engineer
Raul Monzolo:
Open PCR Machine Engineer
Johnny Montez:Open PCR Machine Engineer
Johnny Montez:
Open PCR Machine Engineer
Robert Sanchez:Research and Design Specialist
Robert Sanchez:
Research and Design Specialist
Group 1
Group 1


LAB 2 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green dye is a fluorescent dye that binds to double-stranded DNA, but will stain single-stranded DNA with lower performance.

Single-Drop Fluorimeter
Fluorimeter is a device that is used to measure the amount fluorescence in a single drop.

Image:Photo(2).JPG


How the Fluorescence Technique Works
[In your own words, a summary of the information from page 9 of the worksheet]



Procedure

Smart Phone Camera Settings

  • We used an iPhone 5
    • Flash:Off
    • ISO setting:NA
    • White Balance:NA
    • Exposure:NA
    • Saturation:NA
    • Contrast:NA
  • No additional phone used
  • A time delay setting was used, allowing 5 seconds for the light box to be closed before an image was captured.

Calibration
In order to measure unknown concentrations of DNA with the fluorimeter by measuring fluorescence, we must first measure the fluorescence of known concentrations of DNA using the same technique. To accomplish this, we used an assay of several known concentrations of calf Thymus DNA in conjunction with SYBR green dye. We took pictures of the fluorescing drops and used imaging software to measure the fluorescence. SYBR green dye fluoresces green and the ImageJ software allows the colors of one picture to be split into pictures of the component colors. We can then select just the green light and measure the pixel density, allowing us to quantify the fluorescence. With a known concentration and a known pixel density, we can then define a relationship between the two which will allow us to later measure the fluorescence of an unknown sample to determine the DNA concentration of it.


We inserted the iPhone inside a cradle that gave us a right angle view. Afterward we adjusted the height of the phone so that it is as even with the drop on the slide.

Image:Photo(1).JPG
The image above shows the set-up used. The camera phone in the fore ground is focused on the large drop of dye and sample on the fluorimeter behind it. The drop is lit by the blue LED light and the rest of the fluorimeter is darkened by the surrounding light box

  • Distance between the smart phone cradle and drop = 3cm



Solutions Used for Calibration

Calf Thymus DNA solution (microg/mL) Volume of DNA Solution (μL) Volume of SYBR GREEN I Dye solution (μL) Final DNA concentration in PicoGreen Assay (ng/mL)
0 80 80 blank
.25 80 80 .125
.5 80 80 .25
1 80 80 .5
2 80 80 1
5 80 80 2.5



Placing Samples onto the Fluorimeter

  • Align light between first two clean rows of spots so it will shine directly at the drop, secure slide with tightening screw
  • Using a clean micro-pipette tip, slowly place 80μL of SYBR green dye allowing it to span between both spots
  • Using a clean micro-pipette tip, add 80μL of the sample solution to the SYBR dye
  • Complete assembly by placing the camera/cradle in position and cover with light box
  • Start timed delay picture on phone camera
  • Close light box quickly and carefully, then wait the allotted time for the picture to complete
  • Extract and discard solution from the slide

These steps were repeated in triplicate for each of the samples listed above.


Data Analysis

Representative Images of Samples


Image J Values for All Samples [See worksheet page 5]

Concentration Integrated Density Integrated Density of Background Area of Circle Mean Gray Value
Water213454??669131.902
Water93371??242238.551
Water65949??121954.101
0.25593913??733380.992
0.25569386??698881.481
0.25791593??891788.773
0.5651140??777483.759
0.5453045??514688.038
0.52142033??15458138.571
12458883??12175201.962
11836054??9380195.741
12223630??11335196.174
21721563??6868250.664
23215604??12780251.612
22577701??10205252.592
53957423??15704252.001
54194978??16748250.476
55088859??20200251.924

[Add more rows as needed]


Fitting a Straight Line




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