SYBR Green Dye SYBR Green Dye is a dye that is used as a nucleic acid stain in PCR and other biological and biochemistry procedures. The dye binds to DNA, which causes it to absorb the blue light emitted by the fluorimeter and to radiate a green light. The dye can bind to double stranded DNA the best, but can also bind to single stranded DNA and RNA. In this lab, it is used to quantify the DNA that resulted from the PCR.
Single-Drop Fluorimeter The fluorimeter is a small, black box with a part on top of it where the slide can slide in. It has a light switch to turn on the blue light, which shines through the sample that is put on the slide. There is a dial on the left side helps adjust the light so that it shines through the sample. It also comes with a stand where you can put your phone to take pictures of the sample and a file box to cover it while the picture is being taken.
How the Fluorescence Technique Works The technique works by adding the SYBR green dye and seeing how much green light is emitted by the sample. The more green light that is emitted, the higher the concentration of DNA is. The slides that are put into the fluorimeter have a particular surface that allows the drops of sample to remain spherical. This allows the blue light to shine through the sample and emit the green light.
Procedure
Smart Phone Camera Settings We used an Apple iPhone 4 to capture the images.
Flash: Off
ISO setting: NA
White Balance: NA
Exposure: NA
Saturation: NA
Contrast: NA
Calibration
To set up the camera in front of the fluorimeter, we placed it close enough so that it could focus on the sample. We had to angle it right in order to catch the green light in the picture. To do this, we used plastic plates to raise the fluorimeter so that the drop was aligned with the camera. We placed bubble wrap in front of the camera to keep it from sliding down the camera tray. We also used a black file box around the fluorimeter while taking the picture so that the light would show up better in darkness.
Distance between the smart phone cradle and drop = 4 centimeters
Solutions Used for Calibration[Instructions: See worksheet page 6.]
0ug/mL DNA
0.125ug/mL DNA
O.25ug/mL DNA
0.5ug/mL DNA
1ug/mL DNA
2.5ug/mL DNA
0ug/mL DNA
0.125ug/mL DNA
O.25ug/mL DNA
0.5ug/mL DNA
1ug/mL DNA
2.5ug/mL DNA
0ug/mL DNA
0.125ug/mL DNA
O.25ug/mL DNA
0.5ug/mL DNA
1ug/mL DNA
2.5ug/mL DNA
'
Placing Samples onto the Fluorimeter
1: Place the slide in the fluorimeter.
2: Place 80 uL of the SYBR Green Dye in the middle of the two dots in the center of the rough superhyrdrophobic side of the slide.
3: Place 80 uL of the given calibration DNA concentration sample on the same bead."'
4: Set the timer of the camera and place the shroud over the fluorimeter before the picture is taken.
BME 100 Fall 2013
