LAB 5 WRITE-UP
SYBR Green Dye
SYBR green dye is a molecular dye that fluoresces in the presence of double stranded DNA, but fluoresces weaker in the presence of single stranded DNA. The dye stains the dsDNA produced through polymerase chain reaction (PCR).
The single-drop fluorimeter device detects and measures fluorescence by identifying and quantifying specific fluorescent molecules in a medium. The quantity of fluorescence is directly related to the amount of fluorescent material as well as the amount of molecule that is being detected. Below is an image of the single-drop fluorimeter device.
How the Fluorescence Technique Works
A droplet of the material is placed on the superhyrophobic slide and is exposed to two light beams that decrease the surrounding noise. The fluorescent molecules generate light through excitation by shining light of a shorter wavelength. The energy is released in the form of a photon; the amount released will determine the photon's wavelength and the color of fluorescence which is specific to the SYBR green dye.
Smart Phone Camera Settings
We used an application for the camera and the timer on the iPhone and all of the settings were auto.
- Distance between the smart phone cradle and drop = 10 cm
- Distance between the smart phone camera to drop = 9 cm
[Instructions: See worksheet page 6.]
The picture above shows the distance at which the smart phone was placed from the fluorimeter.
The image above shows the entire setup under the black box while the samples were being photographed.
| Calf Thymus |
| Volume of the |
| Volume of the |
| Final DNA |
SYBR Green I Assay
| 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
To be ensure the samples placed on the slide would result in the correct calibration, the fluorimeter had to be set up properly. The slide was placed onto the fluorimeter and adjusted so that the line of blue light fell directly in between the first two rows of dots. After setting up the fluorimeter, the micro-pipetter was calibrated to 80 microliters and then attached to a clean tip. The tip was inserted into the SYBR Green microtube and then transferred to the slide in between the first two dots so that the blue light illuminated the drop. The contaminated tip was then thrown away. Using a fresh tip still at 80 microliters, the 0 concentration calf thymus was transferred to the slide on top of the already existing SYBR Green drop. The tip contaminated with calf thymus was also thrown away. After aligning the camera on the same plane as the slide and the blue light was directly in line with the drop, a black box was put over the fluorimeter and camera to ensure the blue light was the only light source. The micropipetter then transferred the drop on the slide into the red Dixie cup for proper waste disposal. This process was repeated five times with 0.25, 0.5, 1, 2, and 5 concentrations respectively. Each time, however, the positioning of the drops changed each time, using the next pair of dots with the blue light instead of just sticking with the first two rows the whole time.
Representative Images of Samples
This is an image where a circle was drawn around the droplet with the freehand tool for a sample with no DNA
This is an image where a circle was drawn around the droplet with the freehand tool for a sample with DNA
Image J Values for All Samples
|Calf Thymus DNA Concentration (FINAL), ìg/mL
||Mean Pixel Value
||RAWINTDEN - BACKGROUND
||Subtracted drop & Background
| 5||image 1||4748||237.792||33.446||1129037||10461||1118576
| 5||image 2||4154||242.575||28.777||1007657||9035||998622
| 5||image 3||4868||236.993||37.511||1153682||11881||1141801
| 2||image 1||11985||205.929||67.102||2468057||18891||2449166
| 2||image 2||13688||197.781||68.269||2707229||21565||2685664
| 2||image 3||12756||199.634||67.639||2546535||19279||2527256
| 1||image 1||5265||176.592||67.582||929758||5882||923876
| 1||image 2||3852||128.091||66.473||493406||5122||488284
| 1||image 3||5778||214.353||48.057||1238529||6692||1231837
| 0.5||image 1||13329||108.699||58.687||1448846||18442||1430404
| 0.5||image 2||13674||121.774||52.412||1665140||15932||1649208
| 0.5||image 3||12059||119.689||52.823||1443330||22226||1421104
| 0.25||image 1||4816||52.223||63.274||251508||5646||245862
| 0.25||image 2||5052||32.584||49.664||164615||6841||157774
| 0.25||image 3||5336||39.38||58.745||210133||6694||203439
| 0||image 1||13845||52.334||71.433||724565||14662||709903
| 0||image 2||11992||66.351||75.566||795687||12082||783605
| 0||image 3||13088||44.895||62.211||587588||23493||564095
Fitting a Straight Line
The graph above shows the DNA concentration of the drops for every data point. There were three different data points per concentration.
As we were analyzing our data, we found out that our pictures were not calibrated correctly. Therefore, we decided to calibrate all of the data by hand on our graph. Once we had our data values such as the standard deviation, we multiplied the standard deviation by 2 because we wanted to find our error bar range. The averages and the error bars are shown in the graph above.