BME100 f2013:W1200 Group12 L5

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Group 12

Name: Kevin Lam Role: Background Information
Name: Jacqueline Pauly Role:Image J Measurments
Name: Antonio Moran Role: Took pictures and worked on Procedure section.
Name: Brandon Bartels Role: Worked on procedure section and took a couple of the photos.
Name: Robel Yoseph Role: Background Information and Photographer


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR green dye is a small molecular dye that is used to stain nucleic acids. It fluoresces very well in the presence of double stranded DNA but fluoresces very weakly in water or with single stranded DNA, and is used to detect polymerase chain reactions. During a polymerase chain reaction, SYBR green binds to the double stranded DNA and quantifies non-specific and specific PCR products. In order to be used correctly, SYBR green dye must be calibrated before any tests can be made using it. When being used in tests, the dye will bond to DNA strands emit a green light when it is exposed to blue light, but when exposed to white light the effects are washed out and no visible changes can be seen.


Single-Drop Fluorimeter

Single-Drop Fluorimeter
The Single-Drop Fluorimeter is an instrument that detects the amount of fluorescence in test samples. The fluorescence that is measured has a proportional relationship to the amount of florescent material and, less directly, proportional to the amount of the molecules that are being detected. The amount of molecules present is related to the amount of fluorescent material that is there. The device is made up of a small plastic box with a slot on top for slides with samples to be placed upon it. On the right hand side of the device is a switch that controls the LED light. The slot that lies along the top of the device runs the entire length of the box and allows for the blue LED light to shine across the samples on multi-welled slides. In the picture to the left, the switch on the right of the fluorimeter is the power switch. On the top of the fluorimeter is a place to put the slides. And the glowing blue LED light to the right of the slide is what illuminates through the sample.



How the Fluorescence Technique Works
First, place the slide with the smooth side down into the fluorimeter slot. Adjust the height in order to make sure the camera positioning is right. Place 80 uL of the SYBR green dye between the two circles on the slide. Then add 80 uL of the DNA solution on top of where the green dye was placed. Adjust the slide so that light passes through the middle. Set the timer on the camera until it is focused and then block any external light sources by lowering the flap. Once a picture is taken, it can be uploaded to ImageJ. After everything is done with, the dyes can be disposed of in a proper waste container.




Procedure

Smart Phone Camera Settings


  • Type of Smartphone: iPhone 4s
    • Flash: None
    • ISO setting: Default
    • White Balance: Default
    • Exposure: Default
    • Saturation: Default
    • Contrast: Default


  • Type of Smartphone: iPhone 5
    • Flash: None
    • ISO setting: Default
    • White Balance: Default
    • Exposure: Default
    • Saturation: Default
    • Contrast: Default


Calibration:

The fluorimeter was placed on top of three plates so that it was level with the hight of the iphone's camera. This was because the phone needed to be able to take a picture of the side of the drop. The phone was placed on a stand in front of the fluorimeter and the camera on the phone was about 7 cm away from the drop. This was because the camera needed to be at least 4 cm away from the fluorimeter in order to prevent a blurry photo, and we wern't able to get the camera any closer to the fluorimeter because of the necessary stands underneith the fluorimeter.

PhoneSetup.jpg

  • Distance between the smart phone cradle and drop = 7 cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (microg/mL) Volume of the 2x DNA solution (uL) Volume of the SYBR GREEN Dye solution (uL) 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


Placing Samples onto the Fluorimeter

  1. Take 80uL of SYBR Green I solution and place it on the first two clear circles in the middle of the slide.
  2. Discard tip.
  3. Take 80uL of sample and add it to the drop. It should now contain 160uL, and the sample will be diluted by half.
  4. Discard Tip.
  5. Place slide on the fluorimeter.
  6. Take Photo.
  7. To place another drop on the slide, first remove and discard the 160uL of diluted sample, and discard tip.
  8. Then repeat the first steps, but on the second two clear circles in the middle of the slide.


Data Analysis

  • Image of drop with DNA: positive signal (left picture)
  • Image of drop without DNA: negative signal (right picture)

Drop.JPEG NODNA.JPEG


Calf Thymus DNA Concentration (FINAL), μg/mL Robel ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 image 1 17148 85.963 1474099 44312
2.5 image 2 14596 96.033 1401700 30629
2.5 image 3 16380 87.129 1427177 45502
1 image 1 28428 76.255 2167784 201558
1 image 2 31140 76.532 2383215 86646
1 image 3 32450 64.967 2108191 96341
0.5 image 1 27960 49.391 1380983 67206
0.5 image 2 32308 58.034 1874952 116477
0.5 image 3 31289 68.453 2141825 149224
0.25 image 1 31316 55.766 1746363 76336
0.25 image 2 29292 56.421 1652697 70746
0.25 image 3 26800 60.862 1631094 72910
0.125 image 1 1860 29.625 553404 143432
0.125 image 2 20084 34.631 695536 98156
0.125 image 3 21392 28.595 611711 50055
0 image 1 2163 13.675 29579 11295
0 image 2 3024 13.947 30042 12432
0 image 3 2589 12.056 31003 11982


Calf Thymus DNA Concentration (FINAL), μg/mL LT ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 image 1 3213 32.265 119732 10223
2.5 image 2 2988 36.342 108590 9800
2.5 image 3 1752 20.894 176767 6312
1 image 1 2510 43.255 108571 9005
1 image 2 1904 50.73 100648 6899
1 image 3 2541 44.605 113341 9387
0.5 image 1 2278 44.776 101999 10902
0.5 image 2 2648 45.679 120959 12005
0.5 image 3 2799 46.907 131293 13477
0.25 image 1 2489 58.927 146670 13081
0.25 image 2 2873 64.187 184409 13731
0.25 image 3 2934 62.008 181931 13722
0.125 image 1 2320 15.488 35932 11337
0.125 image 2 1987 15.38 30561 10794
0.125 image 3 1892 16.489 31197 9357
0 image 1 2034 12.473 15760 10345
0 image 2 2546 14.032 31002 115587
0 image 3 2143 12.591 24902 19543
  • Faulty SYBR Green Dye may have caused the results to be abnormal.

Graph LT.JPEG Graph Robel.JPEG