BME100 s2014:T Group6 L5
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LAB 5 WRITE-UP
SYBR Green Dye
How the Fluorescence Technique Works
Smart Phone Camera Settings
Solutions Used for Calibration
Placing Samples onto the Fluorimeter
2.) Second, place 80 microliter drop of SYBR Green 1 on the slide then place 80 microliter of the sample over the SYBR Green.
3.) Third, take a photo of the fluorimeter after lowering the lid of the box, so that it removes as much of stray light.
4.) Finally, remove the box without moving the smartphone and record the sample. Repeat for all samples.
Representative Images of Samples
Image J Values for All Samples
PCR Results Summary
A polymerase chain reaction (PCR) was utilized to amplify the DNA from 2 patients identified here as 46296 and 16946 being screened for point mutations in chromosome 6’s small ubiquitin-like modifier 4 gene (SUMO4). A positive and negative control were amplified alongside the patient’s DNA. PCR results were analyzed by using SYBR Green and a blue light emitting diode (LED) capable of causing the SYBR to fluoresce if DNA was present in the sample.
Before the patient samples could be analyzed, known concentrations of calf thymus DNA had to be used in conjunction with the SYBR to create a curve fit correlating DNA concentration to the intensity of SYBR fluorescence. Since the calf DNA incorporates SYBR in the same manner that human DNA does, the result will be a tool capable of determining the unknown concentration of DNA in the patient PCR tubes.
An iPhone 5 was used to take snap shots of 160 μL droplets, consisting of 80 μL of calf thymus solution that ranged from 0 – 5 μg/mL and 80 μL of SYBR Green. The photos were imported into Image J, a public, java based program available through the National Institute of Health (NIH). Image J has a function that calculates the integrated pixel density of image samples. The integrated density (RAWINTENDEN) of the samples and the known DNA concentrations were incorporated in Graph 1 above.
Each of the 100 μL post PCR human DNA samples were transferred into separate tubes containing 500 μL of buffer solution. The procedure used during calibration was then repeated using the PCR samples in place of calf thymus DNA. The slope intercept equation for the linear trend line established through the calibration procedure was then used to determine the concentrations of human DNA in the sample tubes.
The images for both patients were somewhat dark and opaque with a low intensity of fluorescence. The patient DNA droplet images were both similar to the sample image of the droplet containing no DNA shown above. This qualitative result combined with the relatively low concentration of DNA found by curve fit demonstrates that both patients should be negative for the SUMO4 point mutation.
-Positive control PCR result: 0.186 μg/mL
-Patient 16946: PCR result: 0.029 μg/mL.
-Patient 16946: Negative