LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green Dye is used for the detection of polymerase chain reactions. SYBR Green binds to the double stranded DNA formed during PCR, and quantifies specific and non-specific PCR products. It is a molecular dye that fluoresces in the presence of dsDNA, and fluoresces weakly in the presence of water or single strands of DNA.
A Single-Drop Fluorimeter is a device that measures and detects the amount of fluorescence. The value of fluorescence measured is proportional to the amount of molecules being measured, and is related to the amount of fluorescent material that is present. The device consists of a small box made of plastic that has a rectangular slot on top where the glass slide can be held in place. The blue light is placed on side of device that shines over the glass slide in order to pass through the drop of DNA solution and SYBR Green Dye to measure the fluorescence.
The picture displays the actual Fluorimeter with the hydrophobic slide, camera phone, camera stand, and also the black box located behind the device in the picture that is placed over the device when taking pictures to ensure there isn't exposure to outside light.
How the Fluorescence Technique Works
The first step is to turn on the Fluorimeter, and place it on a flat surface such as a table or lab bench. Then place the glass slide with the smooth side down into the slot on top of the Fluorimeter, once this is completed adjust the height to get the camera placement correct where the slide view is nearly edge-on. After setting up the device, place the 80 uL drop of SYBR Green solution between the first two circles in the middle of the slide. After adding the 80uL of SYBR Green, add 80uL of DNA solution on top of the green dye. Once both solutions are on the slide adjust the slide so that the light passes through the center and to the other side. The distance between the camera and Fluorimeter should be greater than 4cm, if so you can then cover the device with black box keeping one flap up until camera is ready. Set the timer on camera and once it is focused lower the flap to block out the outside light before the picture is taken. Now the data is recorded and ready for analysis on ImageJ, so remove the 160uL drop from the slide and discard in proper waste container.
The SYBR Green dye works as an indicator for the presence of DNA because it only displays fluorescence in the presence of double stranded DNA molecules. In the vials where PCR was successful, higher concentrations of DNA should result in more expression of the SYBR Green fluorescence.
Smart Phone Camera Settings
- Type of Smartphone:Android
- ISO setting:800
- White Balance:Auto
- Exposure: High
- Saturation: Default
- Contrast: Default
Steps for Calibration
1.Turn on the blue light using the switch located on the side of the device
2.Place the phone cradle on table so that it is at least 4 cm from the drop on the Fluorimeter
3.Set phone settings to the proper ones as shown above
4.Focus camera on drop and make sure it is sharply focused before taking the picture.
- Distance between the smart phone cradle and drop = 6cm
Solutions Used for Calibration [Instructions: See worksheet page 6.]
| 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
- Place slide on to the Fluorimeter.
- Place 80 microliter drop of SYBR Green I in between the first two rows of slide by using a pipette.
- Place 80 microliter of the calf thymus solution on top of the SYBR Green I Dye.
- Align the drop so that the LED passes through to the black fiber optic fitting on the other side of the Fluorimeter.
Representative Images of Samples
No DNA Signal Picture
DNA Positive Signal Picture
Image J Values for All Samples
For trials 2 and 5 we used a differant SYBR green dye. This resulted in our numbers be differant because the second dye that we used produced better results. However, the slope is still similar in comparison to trials 0-1 because the rate of increase is still similar. It shows the same results, but when comparing the data a % differance must be accounted for.
Fitting a Straight Line
There are two different graphs for the data due to the use of different SYBR Green Dyes used in the 0-1 concentrations, and the 2-5 concentrations.