BME100 f2013:W1200 Group18 L5

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OUR TEAM

Name: Victoria Platt
Name: Alexandra Olson
Name: Carlyn Harris
Name: Nikhil Patel
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
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
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)
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

[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
No dna screenshot.PNG


Positive DNA Sample ImageJ
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
2.5 image 1 89060 179.093 15950050 320937 15629113
2.5 image 2 89060 179.497 15986030 339696 15646334
2.5 image 3 89568 181.741 16278192 327561 15950631
1 image 1 87704 119.997 10524228 299255 10224973
1 image 2 87704 118.463 10389645 290457 10099188
1 image 3 86532 122.227 10576544 294522 10282022
0.5 image 1 89060 83.038 7395353 306321 7089032
0.5 image 2 89060 81.81 7285978 310260 6975718
0.5 image 3 87136 82.567 7194552 298399 6896153
0.25 image 1 115401 36.066 4508207 371408 4136799
0.25 image 2 112840 38.799 4378129 367704 4010425
0.25 image 3 114532 39.678 4544446 367107 4177339
0.125 image 1 101992 21.448 2187513 316627 1870886
0.125 image 2 99322 21.838 2168998 324564 1844434
0.125 image 3 95148 21.823 2076421 333752 1742669
0 image 1 119632 11.308 1352779 365979 986800
0 image 2 93815 13.139 1232654 365781 866873
0 image 3 835362 12.508 1044850 340181 704669


Fitting a Straight Line

Graph.PNG