SYBR Green Dye
SYBR Green Dye is a membrane permeable nucleic acid dye used in molecular biology labs. The green dye can be placed in a sample with possible DNA and will glow a bright green if DNA is present. This is the most common use of the dye and helps qualitatively analyze if DNA is present in a substance. Although the DNA used in our samples was double stranded DNA, the SYBR Green Dye can also be used to detect single stranded DNA because it has such a high affinity. Although it was placed in the samples after being in the PCR machine, it also can be placed inside a PCR mixture and this allows the dye to bind to the single stranded DNA before it is synthesized by the Taq Polymerase. However, the result is the same. Either way it binds to DNA and glows when DNA is present.
Single-Drop Fluorimeter
Single-Drop Fluorimeter
The Single-Drop Fluorimeter consists of a small, simple plastic box with a apparatus situated on the top where the multi-welled slide can fit in. It has a switch located on the right hand side that allows for the LED light to be turned on or off. The slide of the fluorometer is align so that the blue LED shines through a drop placed over two middle wells. The slide has holes along it to allow for a sample to be placed in them.
How the Fluorescence Technique Works
The fluorometer has a slide with small holes where drops of sample DNA were placed. The drops maintain their round shape because there is a Teflon layer on the slide. The Teflon gives the slide texture and allows the sample drop to maintain its "drop" shape. The reason this is necessary is because the light from the fluorometer can 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.
Procedure
Smart Phone Camera Settings
Setup for Calibration
iPhone 4s
Flash: Off Mode
ISO setting: N/A
White Balance: N/A
Exposure: N/A
Saturation: N/A
Contrast: N/A
Calibration
In order to set up the calibration, the fluorimeter was placed inside a black box. A slide was slid into the fluorimeter where the drops of DNA sample will be placed. The phone cradle was then placed about 11 cm from where the drop was placed on the slide. From there, the sample of DNA was placed on the slides using a pipet. The smart phone was then placed on the cradle, the lid of the black box was closed, and a picture was taken of the drop of DNA sample. This process was repeated with every DNA sample.
Solutions Used for Calibration
Calf Thymus DNA solution concentration (micro/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR GREEN I Dye solution (μL)
Final DNA concentration in PicoGreen 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
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Placing Samples onto the Fluorimeter
Align the slide with the blue LED, so that the light passes in the middle of two rows of wells and reaches the middle of the opposite side.
Place 80 μL of SYBR GREEN in-between two wells.
Next, add 80 μL of DNA sample in the drop that is formed from step #2.
Cover this created sample from light in order to visualize the florescence.
BME 103 Spring 2013
