BME100 f2013:W1200 Group2 L5

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Contents

OUR TEAM

Name: Prycilla Jones
Name: Prycilla Jones
Name: Emily Mei
Name: Emily Mei
Name: Terri Bivins
Name: Terri Bivins
Name: Chandler Varone
Name: Chandler Varone
Name: Amber Bengson
Name: Amber Bengson


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR green dye is a molecule that binds or stains a sample, in the case of this activity a double-stranded DNA or dsDNA. The SYBR green dye will give off a green colored-light when it is illuminated by a blue led light coming from the fluorimeter. This illumination reflects or signals that dsDNA is present in the sample and results to a positive signal; no green illumination means no dsDNA was present and therefore a negative signal.


Single-Drop Fluorimeter
The single-drop fluorimeter is a device that detects fluoresces. It includes a slide that has a smooth surface and a rough surface which is composed of craters that make it a superhydrophobic surface. Due to this superhydrophobic surfave, when the drop is placed on the rough side, the drop tends to have a beach ball or spherical shape which makes it easier to observe the signal. The device also includes a blue LED which excites the SYBR Green 1 in the drop.
Image:Bme35.jpg]


How the Fluorescence Technique Works
The fluorescence technique works by focusing a beam of blue LED light on the sample drop where the blue light, which has a short wavelength but high energy, increases the intensity and excites the SYBR Green 1 in the drop. When the blue light is focused on the drop, the drop absorbs the blue light and reflects or gives off a green colored light or signal.

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: No Flash
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto
  • Type of Smartphone: Samsung Galaxy S3
    • Flash: No Flash
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: 0
    • Saturation: Auto
    • Contrast: Auto


Calibration

  1. Place the phone in the provided plastic cradle.
  2. Adjust the position of the cradle with the phone so that the camera lines up with the center of the fluormeter where the illuminator is present.
  3. If possible, focus the camera on the position of where the droplet would rest.
  4. Measure the distance between the camera and where the droplet would be positioned.
  • Distance between the smart phone cradle and drop = 7.8cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (microg/mL) Volume of the 2X DNA solution (uL) Volume of the SYBR GREEN I 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. Adjust the micropipette using the volume adjustment knob so that it collects 80uL total in volume.
  2. Attaching a new tip, collect 80uL of the SYBR green dye.
  3. Release the SYBR green dye collected in between the two clear circles on the slide.
  4. Dispose the tip and attach a new tip.
  5. With the new tip, collect 80uL of a single concentration of the DNA sample and release the sample above the SYBR green dye so that the solutions mix.
  6. Once the droplet is aligned with the illuminator of the fluorimeter, put the lightbox over with one flap up.
  7. When the camera is refocused and the timer is fixed, start the camera and close the opened flap.
  8. Repeat steps 2-7 two more times for one concentration.
  9. Repeat the whole procedure for all concentrations. This will result in three tests of each concentration of DNA sample.


Data Analysis

Representative Images of Samples


Image:Representative_photo_with_no_dna.JPG

Negative signal, 0 concentration of Calf Thymus DNA


Image:Representative_photo_with_dna.JPG

Positive signal, 1 concentration of Calf Thymus DNA


Image J Values for All Samples


Calf Thymus DNA

Concentration (Final) (microg/mL)

Image Area Mean Pixel

Value

RAWINTDEN

of Drop

RAWINTDEN of

Background

0image 110095218.3313429306271728
0image 27217251.8347371787110135
0image 36841623.1065314104720653
0.25image 17126437.14055178417115164
0.25image 26261636.34453065720279
0.25image 37803646.208717713434677
0.5image 17008452.4377166447183541
0.5image 27253680.93311607404133669
0.5image 37090846.392655711521962


Fitting a Straight Line



Image:Capture_graph_lab_wite_up_5.JPG‎



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