BME100 f2013:W1200 Group18 L5

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Contents

OUR TEAM

Name: Victoria Platt
Name: Victoria Platt
Name: Alexandra Olson
Name: Alexandra Olson
Name: Carlyn Harris
Name: Carlyn Harris
Name: Nikhil Patel
Name: Nikhil Patel
Name: Matthew Armas
Name: Matthew Armas


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is used as a nucleic acid stain; it binds to DNA and absorbs blue light wavelengths while emitting green light wavelengths. While fluorescing very well in the presence of sDNA (double-stranded DNA), RNA and single stranded DNA found in the ending solution of PCR is still highlighted, but not at such a high performance level. Water molecules are not highlighted at all; water containing SYBR Green Dye can be used as a negative control when testing the quantitative results of a PCR machine. Having such a consistent outcome makes SYBR Green Dye the best reagent to use when checking the efficiency of a PCR machine because only completely copied double strands of DNA will be highlight during the use of the fluorimeter (the machine that emits blue light). The brighter the green color of a drop when placed on a fluorimeter, the greater the number of double-stranded DNA present in the sample.


Single-Drop Fluorimeter A fluorimeter is a device used to measure the intensity and wavelengths of fluoresce through the use of light. It has two detectors, one that measures how much light is absorbed and one that measures how much light is emitted. The fluorimeter itself is a black box that has an area on the top of it to place a glass slide with a sample and a horizontally placed blue LED light facing the placement of the slide (as shown below). The device includes a small box that has multiple wells and a glass plate where the sample is placed. There are two LED lights on either side of the sample that shine a blue LED through the sample.

Fluorimeter Set-Up
Image:Flourimeter.jpg


How the Fluorescence Technique Works
Fluorescence technique uses the emission of light due to a molecule's absorption of a shorter wavelength to create an image. In a Single-Drop Fluorimeter, blue light (wavelength value of ~475nm) is emitted from an LED in order to excite the molecules of the SYBR Green Dye that have bonded with the double-stranded DNA in the drop of solution taken from the PCR machine at the end of a completed cycle. Once the blue light is absorbed by the slide sample that has been placed on the fluorimeter, a green light is reflected by the sample (wavelength of ~510nm). This process is essentially what fluorescing is: absorbing one wavelength of which a solution is being exposed to and emitting another higher wavelength. Fluorimeters are intended for assisting in the detection of fluorescence.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 4S
    • Flash: OFF
    • ISO setting:N/A
    • White Balance: N/A
    • Exposure:N/A
    • Saturation:N/A
    • Contrast:N/A


Calibration

Fluorimeter and Camera Setup
Image:Flourimeter with camera.JPG
The camera was positioned in the cradle so that it was standing upright at a 90 degree angle. The cradle and camera is supposed to be at distance greater than 4 cm away from the fluorimeter. In this experiment, it was approximately 5.9 cm away. The camera was also level with drop and focused on it. During the experiment, a picture was taken every three seconds, for a total of three pictures per drop.

  • Distance between the smart phone cradle and drop = 5.9 cm


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

Calf Thymus DNA solution concentration (microg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I Assay (ng/mL)
580802.5
280801
180800.5
0.580800.25
0.2580800.125
08080blank

[Add more rows as needed]


Placing Samples onto the Fluorimeter

  1. Step one, insert the glass slide into the fluorimeter with the rough side facing up.
  2. Step two, turn on the fluorimeter and adjust the slide so that the light emitted from the fluorimeter passes between two sets of holes.
  3. Step three, use the micropipette to drop 80µL of SYBR GREEN I on the slide so that the light travels through the drop.
  4. Step four, use the micropipette to add 80µL of the first of six different concentrations of the DNA solution to the drop of SYBR GREEN I.
  5. Step five, use the micropipette to remove the liquid on the slide after the experiment.
  6. Step six, adjust the slide in the fluorimeter so that its light passes between two new sets of holes.
  7. Step seven, repeat steps three through six, but remember to change the concentration of the DNA solution.


Data Analysis

Representative Images of Samples


No DNA Sample ImageJ
Image:No dna screenshot.PNG


Positive DNA Sample ImageJ
Image:Dna snapshot.PNG


Image J Values for All Samples



Calf Thymus DNA Concentration (FINAL), μg/mL Image AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN - BACKGROUND
2.5image 189060179.0931595005032093715629113
2.5image 289060179.4971598603033969615646334
2.5image 389568181.7411627819232756115950631
1image 187704119.9971052422829925510224973
1image 287704118.4631038964529045710099188
1image 386532122.2271057654429452210282022
0.5image 18906083.03873953533063217089032
0.5image 28906081.8172859783102606975718
0.5image 38713682.56771945522983996896153
0.25image 111540136.06645082073714084136799
0.25image 211284038.79943781293677044010425
0.25image 311453239.67845444463671074177339
0.125image 110199221.44821875133166271870886
0.125image 29932221.83821689983245641844434
0.125image 39514821.82320764213337521742669
0image 111963211.3081352779365979986800
0image 29381513.1391232654365781866873
0image 383536212.5081044850340181704669


Fitting a Straight Line

Image:Graph.PNG




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