OUR TEAM
Our group facilitates lab by having one or two members recording the data and write up information in open wetware while the other members perform the experiment. All contributions on lab work is equal throughout the group though it may not appear in the open wetware history of contirbutions due to the way our group has structured our excecution of lab work. All members of the group perform equal duties and contribute to the completion of all lab work.
LAB 5 WRITE-UP
SYBR Green Dye
The SYBR Green Dye is a dye that becomes fluorescent when in the the same vicinity of dsDNA. This dye does not fluoresce very well when mixed with water or a single stranded DNA. SYBR Green is also commonly used with Real Time PCR.
Single-Drop Fluorimeter
The Single-Drop Fluorimeter is a device that recognizes fluorescence, where the amount of florescent material is quantified and is compared to the size of the molecule of interest.
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How the Fluorescence Technique Works
The Fluorescence Technique focuses on using a small molecule of SYBR Green Dye. This molecule binds exclusively to DNA strands and causes them to fluoresce when subjected to visible light. We can qualitatively analyze the amounts of DNA present in our samples by comparing the intensity of the reflected light. There are different dyes that do work with other machines, but in this lab SYBR green dye was used.
Procedure
Smart Phone Camera Settings
- Type of Smartphone: Galaxy S3
- Flash: off
- ISO setting: 800
- White Balance: Auto
- Exposure: +2
- Saturation: N/A
- Contrast: Auto
Only one phone was used for this test.
- Type of Smartphone: N/A
- Flash:
- ISO setting:
- White Balance:
- Exposure:
- Saturation:
- Contrast:
Calibration
Our method of aligning our smartphone was guess and check. The cradle alone would not allow us to line up our phone with the droplet, so we had to come up with something else. We were provided plates, so we placed them under the cradle in order to lift the camera. After testing different heights with plates, we ended up putting 7 plates under our phone cradle and rested the galaxy s3 sideways with the cradle as support.
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- Distance between the smart phone cradle and drop = 9.5 centimeters
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (micro/mL) |
Volume of the 2x DNA solution (uL) |
Volume of SYBR Green I dye (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
- Step One: With gloves on, locate the rough superhydrophobic side of the slide.
- Step Two: Power the fluorimeter and place the slide in with the rough superhydrophobic side facing up.
- Step Three: Place one 80 micro-liter drop of SYBR green dye on the first two circles in the middle column of the slide.
- Step Four: Put one micro-liter drop of the sample solution on top of the existing drop of green dye.
- Step Five: Move the slide so that the light from the fluorimeter is going through the center of the drop.
Data Analysis
Representative Images of Samples
No DNA
With DNA
Image J Values for All Samples
Fitting a Straight Line
|
BME 100 Fall 2013
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