BME100 f2013:W900 Group17 L5

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OUR TEAM

Name: Abrar Bakhsh
Name: Jeremy Becker
Name: Luis Hernandez
Name: Alison Llave
Name: Naaz Maududi

LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green I dye is a cyanine dye that binds to DNA and is commonly used as a nucleic acid stain. The dye absorbs blue light (497 nm) and emits green light (520 nm). While it preferentially binds to double stranded DNA, the dye will also bind to single strand DNA with the reslt being somewhat lower performance. Finally, SYBR Green I dye is commonly used in PCR, gel electrophoresis, and fluorescence microscopy.

Single-Drop Fluorimeter
This device detects DNA based off of fluorescence from a molecule bound to the DNA such as SYBR green dye. The DNA sample that is placed on the slide which is then inserted in the machine and shined with blue LED light. After the light hits the sample, the DNA should emit a dim green glow. To collect data a phone is used to capture the green light.

How the Fluorescence Technique Works
Fluorescence technique works by adding dye to a concentration of DNA, the dye that was used was SYBR Green Dye. The Teflon coated glass will keep the droplets in place.From the single-drop fluorimeter a beam of blue light is emitted. Also a fluorescent green light will be emitted from the droplet which could then be measured by taking a picture on a smart phone and afterwards using Image J to analyze the image.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S3
    • Flash: None
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Set on the highest exposure setting (2.0)
    • Saturation: Not applicable
    • Contrast: Not applicable
  • Type of Smartphone: Motorola Razr HD Maxx
    • Flash: None
    • ISO setting: Not applicable
    • White Balance: Auto
    • Exposure: Set on the highest exposure setting
    • Saturation: Not applicable
    • Contrast: Not applicable


Calibration

A light box was placed over the fluorimeter so that no outside source of light can get in. As seen below, the apparatus must then be set up so that the camera lens of the phone is at a 90 degree angle relative to the droplet. After the image of the droplet on the phone was focused, the distance of the camera lens from the droplet was measured using a ruler. Calibration3.jpg

  • Distance between the smart phone cradle and drop = 5.5 cm for S3 and 9.0 cm for Razr


Solutions Used for Calibration

Calf Thymus DNA Solution (mg/mL) Volume of DNA (μL) Volume of SYBR Green I Dye (μL) Final DNA Concentration (mg/mL)
0 80 80 blank
0.25 80 80 0.125
0.5 80 80 0.25
1 80 80 0.5
2 80 80 1
5 80 80 2.5


Placing Samples onto the Fluorimeter

  1. A glass slide was properly placed with the smooth side down onto the fluorimeter. The first row of the circles is placed in alignment with the blue light of the fluorimeter.
  2. Taking the micropipette, the volume the micropipette will be measuring should be 80 μL.
  3. 80 μL of the SYBR Green dye is placed in the middle of the first row of circles.
  4. 80 μL of 0 mg/mL calf thymus DNA solution is placed on the SYBR green dye drop.
  5. With the phone properly placed at the distance indicated by the calibration and set with a 5-second timer, the black box was placed around the system, as seen in the image below.
  6. Pictures were taken with the black box closed to minimize the amount of light entering the system.
  7. Repeat the above steps with each of the calf thymus DNA solutions, moving down a row of circles each time to prevent cross-contamination.


Calibration5.jpg


Data Analysis

Representative Images of Samples

DNAFluorescence.jpg

Green5.jpg


Image J Values for All Samples

Calf Thymus DNA Concentration (FINAL), ìg/mL Image AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 S3 #1 7296 20.229 147593 596
2.5 S3 #2 5238 24.158 126541 712
2.5 Razr 3544 21.257 75336 378
1 S3 #1 6656 17.198 114468 1726
1 S3 #2 7484 18.79 140624 1019
1 Razr 3954 20.256 80092 3516
0.5 S3 #1 6400 14.445 92450 1932
0.5 S3 #2 6784 16.593 112568 911
0.5 Razr 3644 15.472 56381 794
0.25 S3 #1 6892 10.166 70066 1107
0.25 S3 #2 6840 10.635 72740 1162
0.25 Razr 5507 21.861 120522 9814
0.125 S3 #1 8129 16.839 136886 1428
0.125 S3 #2 6780 13.416 90959 1458
0.125 Razr 314 12.911 4054 402
0 S3 #1 4932 16.067 79241 26636
0 S3 #2 4705 14.977 70465 2123
0 Razr 406 15.099 6130 217


Fitting a Straight Line

CalfThymus7.jpg

Possible Error While the data points taken using the Samsung Galaxy phone had high precision, the data points taken using the Motorola Razr were found to be most likely inaccurate due to the exceedingly low values when compared to the acceptable range of values. This was due, most likely, to the phone camera's low adjustibility in regards to settings and the inferiority of the sensor when compared to the Samsung Galaxy.