LAB 5 WRITE-UP
SYBR Green Dye
SYBR green dye is one of the asymmetrical high-sensitivity reagents for staining DNA and RNA in electrophoretic gels. When combined, the gel stains offer greater sensitivity towards measuring an accurate reading. In this lab, specifically, SYBR green dye is primarily used to stain and dye the nucleic acid, which makes it possible to see large amounts of DNA by the naked eye. Essentially, SYBR green dye specifically fluoresces in the actual presence of double stranded DNA. However, in the presence of the single stranded DNA, the SYBR green dye fluoresces even more so. The SYBR Green Dye absorbs blue light and emits green light, explaining the ability to visualize the fluorescence. Generally, SYBR green dye deliver highly sensitive real time PCR results in moderate timings, allowing lab members to process the information in a shorter period of time.
The single drop fluorimeter is a device that detects and measures fluorescence by specifying certain fluorescent molecules in a given area. The amount measured is proportional to the amount of fluorescent material to the amount of molecule that is detected. More specifically, the Single-Drop Fluorimeter shines blue light through a drop of DNA/SYBR green dye mix which absorbs the blue light making it possible to see the DNA in the drop of liquid.
How the Fluorescence Technique Works
The Fluorescence Technique Works by using the surface of the slide, which is mainly glass, which allow for the blue LED light to focus specifically on the droplet. Because of the difference in the surface, the SYBR green dye is able to bind with the DNA. Once the data is processed, the images will be used to determine the specificities for each of the solutions. The specific the fluorescent technique works because it gives the person the ability to see DNA with the naked eye so that the proof of DNA is apparent.
Smart Phone Camera Settings
- Type of Smartphone: iPhone
- Flash: Flash was inactivated.
- ISO setting: ISO was set at 800.
- White Balance: White Balance was set at auto.
- Exposure: Exposure was set to the highest setting.
- Saturation: Saturation was set to the highest setting.
- Contrast: Contrast was set to the lowest setting.
Place smartphone in phone holder with camera in line with the slide and close enough to get an ideal picture for the experiment.
- Distance between the smart phone cradle and drop =
- 5.75 cm for the blank sample
- 5.75 cm for the .125 final DNA concentration sample SYBR GREEN I Dye
- 5.75 cm for the .250 final DNA concentration sample SYBR GREEN I Dye
- 5.75 cm for the .500 final DNA concentration sample SYBR GREEN I Dye
- 5.50 cm for the 1.000 final DNA concentration sample SYBR GREEN I Dye
- 5.75 cm for the 2.500 final DNA concentration sample SYBR GREEN I Dye
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (microg/mL) || Volume of the 2X DNA solution (μL) || Volume of the SYBR GREEN I Dye solution (μL) || 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
- Pipet 80 μL of SYBR Green solution in the center of the first two rows of the slide which should be lined up with the blue light.
- Discard the discard-able pipet tip and then place a new non contaminated one on.
- Pipet 80 μL of the calf thymus solution being tested at that point and place it in the same drop as the SYBR Green.
- "Align the camera and set a timer on the camera to take three images."
- Place the cover on the box to prevent any additional light from coming in and take a picture of the fluorescence.
- Once the picture is taken pipet the sample off the slide, discard used tip and adjust fluorimeter slide accordingly.
- Repeat the steps above two more times for a total of three images for each concentration.
Representative Images of Samples
Image J Values for All Samples
|Calf Thymus DNA Concentration (FINAL), μg/mL
||Mean Pixel Value
||RAWINTDEN OF THE DROP
||RAWINTDEN OF THE BACKGROUND
| 2.5||Image 1||13985||199.45||13236789||12058723
| 2.5||Image 2||12823||187.45||12789541||11902538
| 2.5||Image 3||11907||175.34||8923587||110926359
| 1||Image 1||6627||189.56||13092679||94561092
| 1||Image 2||7543||192.45||14982673||80268121
| 1||Image 3||6802||162.91||56823||62390123
| 0.5||Image 1||13897||218.82||87652||145820372
| 0.5||Image 2||12096||187.45||99367||18902736
| 0.5||Image 3||14782||175.23||75239||17253820
| 0.25||Image 1||5592||55.62||51725||2679012
| 0.25||Image 2||6921||69.87||50267||3028912
| 0.25||Image 3||7590||74.23||47291||2561021
| 0||Image 1||12679||85.32||1775678||8012719
| 0||Image 2||13792||74.29||1745237||7800191
| 0||Image 3||13694||79.67||1865890||826102
Fitting a Straight Line