OUR TEAM
LAB 5 WRITE-UP
Background Information
SYBR Green Dye
A molecular dye that fluoresces in the presence of dsDNA, however it fluoresces weakly in the presence of water or single strands of DNA.
Single-Drop Fluorimeter
A device that detects and measures fluorescence. The value measured is related to the amount fluorescent material present, and proportional to the amount of molecule being measured.
How the Fluorescence Technique Works
The Fluorescence technique is used when testing for the presence of double stranded DNA in a solution. This technique involves a dye being added to a solution. The color of that dye shows up when certain wavelengths are shone through the solution and the solution has double stranded DNA. For this experiment the SYBR Green I dye is used. This is used because it fluoresces well in the presence of double stranded DNA making it easy to detect whether or not a solution has double stranded DNA. The source of the light that is shone through the solution in this experiment is from a fluorimeter, a device that detects fluorescence.
Procedure
Smart Phone Camera Settings
- Type of Smartphone: Apple iPhone 4s
- Flash: LED (default phone setting for flash)
- ISO setting: Left on default setting
- White Balance: Left default setting
- Exposure: 120 ms
- Saturation: Left default setting
- Contrast: Left default setting
No phone settings were changed. Flash was turned off, so the standard iSight camera settings were used.
Calibration
1. Before placing the camera in the cradle, the flash must be turned off, the ISO must be set to 800 or higher, the white balance must be set to auto, the exposure must be set to the highest setting, the saturation must be set to the highest setting, and the contrast must be set to the lowest setting.
2. Once your camera is set up, place your phone into the provided cradle.
3. Once the phone is set into the cradle, the fluorimeter may need to be adjusted so that the the camera is at eye level with the side of the drop.
4. The cradle with the phone in it must be at least 4 cm apart but may need to be adjusted further apart depending on how close the camera needs to be in order to focus.
5. Once the phone is at a distance where the camera will be able to focus on the drop, measure this distance (between the camera and the light).
- Distance between the smart phone cradle and drop = approximately 5.5 cm
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (microg/ml) |
Volume of the 2X DNA soltuion (microliter) |
Volume of the SYBR GREEN I Dye solution (microliters) |
Final DNA concentration in SYBR GREEN I Assay (ng/mL)
|
| 5 |
80 |
80 |
2.5
|
| 2 |
80 |
80 |
1
|
| 1 |
80 |
80 |
0.5
|
| .5 |
80 |
80 |
0.25
|
| .25 |
80 |
80 |
0.125
|
| 0 |
80 |
80 |
blank
|
Placing Samples onto the Fluorimeter
- Set pipette to 80 microliters.
- Place a tip on the pipette. Measure 80 microliters of the SYBR GREEN I and place in the middle of the first two rows on the slide. Place the now used tip into the trash.
- Place a new tip on the pipette. Measure 80 microliters of one of the calf thymus and place in the middle of the drop. Place the now used tip into the trash.
Data Analysis
Representative Images of Samples
This is an image of the result drop with no green dye.
This is an image of the result drop with green dye.
Image J Values for All Samples
| Calf Thymus DNA concentration (FINAL), microg/ml |
INTDENS Readings Before Background Correction
|
| 5 |
19926294
|
| 2 |
11854900
|
| 1 |
6784819
|
| .5 |
4696140
|
| .25 |
4341330
|
| 0 |
600443
|
Fitting a Straight Line
|
BME 100 Fall 2013
