SYBR Green Dye
SYBR green dye is a small molecular dye that is used to stain nucleic acids. It fluoresces very well in the presence of double stranded DNA but fluoresces very weakly in water or with single stranded DNA, and is used to detect polymerase chain reactions. During a polymerase chain reaction, SYBR green binds to the double stranded DNA and quantifies non-specific and specific PCR products. In order to be used correctly, SYBR green dye must be calibrated before any tests can be made using it. When being used in tests, the dye will bond to DNA strands emit a green light when it is exposed to blue light, but when exposed to white light the effects are washed out and no visible changes can be seen.
Single-Drop Fluorimeter
The Single-Drop Fluorimeter is an instrument that detects the amount of fluorescence in test samples. The fluorescence that is measured has a proportional relationship to the amount of florescent material and, less directly, proportional to the amount of the molecules that are being detected. The amount of molecules present is related to the amount of fluorescent material that is there. The device is made up of a small plastic box with a slot on top for slides with samples to be placed upon it. On the right hand side of the device is a switch that controls the LED light. The slot that lies along the top of the device runs the entire length of the box and allows for the blue LED light to shine across the samples on multi-welled slides. In the picture to the left, the switch on the right of the fluorimeter is the power switch. On the top of the fluorimeter is a place to put the slides. And the glowing blue LED light to the right of the slide is what illuminates through the sample.
How the Fluorescence Technique Works
First, place the slide with the smooth side down into the fluorimeter slot. Adjust the height in order to make sure the camera positioning is right. Place 80 uL of the SYBR green dye between the two circles on the slide. Then add 80 uL of the DNA solution on top of where the green dye was placed. Adjust the slide so that light passes through the middle. Set the timer on the camera until it is focused and then block any external light sources by lowering the flap. Once a picture is taken, it can be uploaded to ImageJ. After everything is done with, the dyes can be disposed of in a proper waste container.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 4s
Flash: None
ISO setting: Default
White Balance: Default
Exposure: Default
Saturation: Default
Contrast: Default
Type of Smartphone: iPhone 5
Flash: None
ISO setting: Default
White Balance: Default
Exposure: Default
Saturation: Default
Contrast: Default
Calibration:
The fluorimeter was placed on top of three plates so that it was level with the hight of the iphone's camera. This was because the phone needed to be able to take a picture of the side of the drop. The phone was placed on a stand in front of the fluorimeter and the camera on the phone was about 7 cm away from the drop. This was because the camera needed to be at least 4 cm away from the fluorimeter in order to prevent a blurry photo, and we wern't able to get the camera any closer to the fluorimeter because of the necessary stands underneith the fluorimeter.
BME 100 Fall 2013
