LAB 5 WRITE-UP
SYBR Green Dye
SYBR green dye is a cyanide dye that is used as a stain for nucleic acid. SYBR Green I binds to DNA. The resulting mixture emits green light and absorbs blue light. The dye binds to double stranded DNA with the highest performance. However, it will also bind to single stranded DNA with a lower performance. It will also bind to RNA but with an even lower performance than single stranded DNA. SYBR Green Dye is used in many experiments in molecular biology along with biochemistry. It is used in some method of quantitative PCR to determine amounts of DNA. It is also used in gel electrophoresis to visualize DNA. SYBR Green Dye is used as a replacement for ethidium bromide, because it is safer to work with and easier to dispose of.
The single-drop fluorimeter device is used to measure and detect fluorescence using the amount of fluorescent material, intensity, and wavelength distribution of emission spectrum. The apparatus consists of a small plastic box with a thin rectangular layer of space on the top where the multi-welled slide can fit in. The slot is present on the device for the glass slide to be placed on. Droplets of the SYBR Green dye and DNA solutions are placed on the glass slide for the light to be passed through. The slide has these holes along it to act as place holders for the DNA samples to be placed on. The box emits a blue light that can be turned on or off by the switch located on the right hand side that allows for the LED light to pass through the sample. The slide of the fluorimeter is aligned so that the blue LED shines through a drop placed over two middle wells.
How the Fluorescence Technique Works
Fluorescence is a very sensitive detection technique that uses the generation of light by a molecule due to excitation by shining light of a shorter wavelength. The fluorescence technique works by adding the fluorescent SYBR dye to a sample with a certain amount of DNA concentration. The fluorimeter has a slide with small holes where drops of sample DNA are placed. Teflon coated glass slides are used in a single-drop fluorimeter in order to produce spherical drops on the surface as a result of the hydrophobic properties created by the various circles of uncovered glass. The drops are able to maintain their round shape on the slide because of the Teflon layer on the slide. The Teflon gives the slide texture and allows the sample drop to maintain its shape. Maintaining this shape is necessary in order for the light from the fluorimeter to go directly through the drop to increase the intensity of the DNA glow. It also allows for the SYBR Green to move to the surface, which allows for the glow to be seen on the top of the drop. All of this makes it easier to observe and analyze whether the sample has a green glow, and whether there was DNA present in the sample.
Smart Phone Camera Settings
- Type of Smartphone: Galaxy S4
- Flash: Off
- ISO setting: 800
- White Balance: Auto
- Exposure: +2.0
- Saturation: Unable to Change
- Contrast: Unable to Change
1. Turn on the blue LED light.
2. Turn on the smart phone's camera and edit the settings, if possible.
- Set ISO to 800 or higher.
- Set white balance to auto.
- Set exposure to the highest setting.
- Set saturation to the highest setting.
- Set contrast to the lowest setting.
3. Place the smart phone on the cradle at a right angle to the slide and adjust the height of the fluorimeter using the plastic trays so that the camera lines up with the sideways drop.
4. Adjust the distance between the smart phone on its cradle and the slide so that it is as close as possible without getting a blurry image. The camera should be 7 cm away from the drop.
5. Use a ruler to record the distance between the smart phone cradle and the drop. Be careful not to move the camera, cradle, or fluorimeter very much, as this will cause the light collected to change slightly if there is a significant difference from one image to the next.
6. Place an 80 microliter drop of SYBR Green I in the middle of the first two rows of the slide using the plastic pipette so that the drop is pinned and spherical. Then add 80 microliters of one of the calf thymus dilutions. This solution is now the full sample, or drop.
7. Align the drop with the blue light so the light shines directly through it.
8. Place the light box over the fluorimeter and camera so that when the front flap is closed, no light reaches the set-up. Set a 5 second timer on the phone's camera so there is time to close the flap before the picture is taken.
9. Take a picture of the drop, making sure it is in focus and that the phone is stationary at all times.
10. Carefully remove the light box without moving the smart phone.
11. Using the pipette, remove the 160 microliter drop from the surface of the slide. With the next sample, use a different part of the slide and adjust the slide's position accordingly. Each slide can be used for up to three samples.
- Distance between the smart phone cradle and drop = 7 cm
Solutions Used for Calibration
| Calf Thymus DNA Solution Concentration (microg/mL) || Volume of the 2X DNA Solution (microliters) || 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
| 0.5 || 80 || 80 || 0.25
| 0.25 || 80 || 80 || 0.125
| 0 || 80 || 80 || BLANK
Placing Samples onto the Fluorimeter
- Place the super-hydrophobic slide into the fluorimeter with the rough side up, in front the the blue LED light.
- Set the micro-pipette to 80 uL and withdraw SYBR Green I, placing it onto the slide on the middle circle in the second row.
- Withdraw 80 uL of the sample solution and place it on top of the SYBR Green I drop.
- Move the slide to center the drop in the light.
Representative Images of Samples
Negative signal image (No DNA)
Positive signal image (with 5 µg/mL of DNA)
Image J Values for All Samples
|Calf Thymus DNA Concentration (FINAL), μg/mL
||Average Mean Pixel Value
||Rounded Average RAWINTDEN of The Drop
||Rounded Average RAWINTDEN of The Background
Fitting a Straight Line