BME100 f2013:W1200 Group12 L5

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Contents

Group 12

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

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.

  • 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)


Calf Thymus DNA Concentration (FINAL), μg/mL Robel ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5image 11714885.963147409944312
2.5image 21459696.033140170030629
2.5image 31638087.129142717745502
1image 12842876.2552167784201558
1image 23114076.532238321586646
1image 33245064.967210819196341
0.5image 12796049.391138098367206
0.5image 23230858.0341874952116477
0.5image 33128968.4532141825149224
0.25image 13131655.766174636376336
0.25image 22929256.421165269770746
0.25image 32680060.862163109472910
0.125image 1186029.625553404143432
0.125image 22008434.63169553698156
0.125image 32139228.59561171150055
0image 1216313.6752957911295
0image 2302413.9473004212432
0image 3258912.0563100311982


Calf Thymus DNA Concentration (FINAL), μg/mL LT ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5image 1321332.26511973210223
2.5image 2298836.3421085909800
2.5image 3175220.8941767676312
1image 1251043.2551085719005
1image 2190450.731006486899
1image 3254144.6051133419387
0.5image 1227844.77610199910902
0.5image 2264845.67912095912005
0.5image 3279946.90713129313477
0.25image 1248958.92714667013081
0.25image 2287364.18718440913731
0.25image 3293462.00818193113722
0.125image 1232015.4883593211337
0.125image 2198715.383056110794
0.125image 3189216.489311979357
0image 1203412.4731576010345
0image 2254614.03231002115587
0image 3214312.5912490219543
  • Faulty SYBR Green Dye may have caused the results to be abnormal.

Image:Graph LT.JPEG Image:Graph Robel.JPEG

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