LAB 5 WRITE-UP
SYBR Green Dye
This is a small molecular dye that fluoresces very well in the presence of dsDNA however fluoresces very weakly in water or with single strands of DNA
The Single-Drop Fluorimeter is the instrument that detects the fluorescence in the drop on the slide based on the amount of fluorescent material.
How the Fluorescence Technique Works
In general, fluorescence occurs when light of a specific wavelength can excite a material resulting in a longer wavelength of light to emit. In this experiment,
high concentrations of DNA are added to SYBR Green 1. The greater the amount of DNA, the more it will fluoresce in the SYBR Green 1 solution. The
optical caustic effect of the drop also causes the intensity of the light to increase on the surface of the drop so that the camera can detect the fluorescence more easily.
Smart Phone Camera Settings
- Type of Smartphone: iPhone 5s
- Flash: None
- ISO setting: N/A
- White Balance: N/A
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
In this experiment, the iPhone 5 was set in a cradle that propped it up so that multiple photos could be taken without moving or adjusting the phone and affecting results. The fluorimeter was placed on various objects in order to raise it to the height level with the camera lens. With this set-up, the drop can be photographed directly from the side which is optimal in order to detect the fluorescence and benefit from the optical caustic effect.
- Distance between the smart phone cradle and drop = 4cm
Solutions Used for Calibration
Placing Samples onto the Fluorimeter
- Set up the fluorimeter such that the smart phone camera will be level with the drop.
- Place the smooth side of the slide face down in the fluorimeter
- Start by putting 80uL of SYBR Green 1 solution centered between the first two clear circles on the slide.
- Now add 80uL of the diluted PCR product solution to the SYBR Green 1 drop.
- Move the slide so that the light lands on the center of the drop.
- Focus the camera on the drop and set the timer so that it will take multiple pictures every few seconds.
- Cover the box and close the front flap making it as dark as possible as the camera takes the pictures.
- Remove the drop from the slide and discard the liquid in the proper waste container. Replace the slide and start the process again for the various concentrations of the PCR product solution.
Representative Images of Samples
Image J Values for All Samples
Fitting a Straight Line
Your positive control PCR result was -0.0028785 μg/mL
Your negative control PCR result was -0.0033691 μg/mL
Patient 1 ID: 56366: Values of -0.0029257μg/mL, -0.0036317μg/mL, -0.0038241μg/mL
Patient 2 ID: 86361: Values of -0.0029509μg/mL, -0.002359μg/mL, -0.0052083μg/mL.
Patient 1 ID# 56366: The results for this patient are consistently more similar to those of the negative control group
Patient 2 ID# 86361: The results for this patient vary, however they are explainable. The first two experiments point
toward the positive control. For the 2R3 experiment, there was a shortage of the SYBR Green 1 solution so it was borrowed
from another group for this section. It had been left uncovered and therefore affected our results.