BME103 s2013:T900 Group1 L2

From OpenWetWare

Jump to: navigation, search
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
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 detection probe that absorbs blue wavelengths of light and emits green wavelengths, enabling it to bind to double-stranded DNA. Although it performs well in the presence of double-stranded DNA, it fluoresces less effectively in water and with single-stranded DNA.

Image:SYBRgreen.jpg


Single-Drop Fluorimeter
A Fluorimeter is a device used to quantitatively measure fluorescent material in a sample or substance in such a way that it is proportional to the amount of molecules being detected.

Image:Photo(2).JPG


How the Fluorescence Technique Works
The components of a fluorimeter are designed specifically to enhance the environment of small, aqueous quantities for fluorescent analysis. The Teflon coated slides have multiple circles of bare glass that exhibit superhydrophobic properties which allow spherical drops to form on top of the glass. When light from the Blue LED is concentrated on the substance, the intensity excites the SYBR green Dye and causes it to fluoresce. As the dye combines with molecules such as dsDNA, it forms a structure that travels to the surface of the drop for observation. SYBR green fluorescence is a reliable technique because its sensitivity reacts favorably in its environment.



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

Concentration Area of Circle Mean Gray Value Raw Integrated Density Raw Integrated Density of Background
Water756633.15325083424944
Water207240.972848955326
Water655312.8918447325857
0.251133575.73385843429401
0.251001078.33378411726530
0.251211683.009100573944131
0.5996479.56979283025061
0.51026078.60480648128115
0.517391137.035238318040816
114512178.775259437754092
118151167.444303926742992
116108175.72283050334749
212623224.804283769927499
215887218.648347366036706
216880223.931377995357381
519160236.711453538441840
519612236.43463685954123
520035240.357481555948482



Fitting a Straight Line


**PLAGIARISM WARNING: A DUPLICATE VERSION OF THIS IMAGE HAS BEEN FOUND ON ANOTHER TEAM'S LAB REPORT**




Personal tools