OUR TEAM
LAB 5 WRITE-UP
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.
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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
- Place the phone in the provided plastic cradle.
- 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.
- If possible, focus the camera on the position of where the droplet would rest.
- 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
- Adjust the micropipette using the volume adjustment knob so that it collects 80uL total in volume.
- Attaching a new tip, collect 80uL of the SYBR green dye.
- Release the SYBR green dye collected in between the two clear circles on the slide.
- Dispose the tip and attach a new tip.
- 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.
- Once the droplet is aligned with the illuminator of the fluorimeter, put the lightbox over with one flap up.
- When the camera is refocused and the timer is fixed, start the camera and close the opened flap.
- Repeat steps 2-7 two more times for one concentration.
- 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
Negative signal, 0 concentration of Calf Thymus DNA
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
|
| 0 |
image 1 |
100952 |
18.331 |
3429306 |
271728
|
| 0 |
image 2 |
72172 |
51.834 |
7371787 |
110135
|
| 0 |
image 3 |
68416 |
23.1065 |
3141047 |
20653
|
| 0.25 |
image 1 |
71264 |
37.1405 |
5178417 |
115164
|
| 0.25 |
image 2 |
62616 |
36.34 |
4530657 |
20279
|
| 0.25 |
image 3 |
78036 |
46.208 |
7177134 |
34677
|
| 0.5 |
image 1 |
70084 |
52.437 |
7166447 |
183541
|
| 0.5 |
image 2 |
72536 |
80.933 |
11607404 |
133669
|
| 0.5 |
image 3 |
70908 |
46.392 |
6557115 |
21962
|
|
|
Fitting a Straight Line
|
BME 100 Fall 2013
