Camera should be set in the cradle at least 4 cm away from the drop placed on the slide
Camera should be set on a timer of three seconds in order to allow for a proper time of closing the box and no outside light to contaminate the pictures
The lens should be level with the drop (all of the drop's spherical surface from top to bottom should be in the picture)
Adjust Fluorimeter height if necessary to get it in the proper view of the phone camera
Distance between the smart phone cradle and drop = 6.8 cm
Solutions Used for Calibration
Calibration via H2O
Calibration via the standard given which was a concentration 0.25 units of DNA per microliter |
Calibration via the standard given which was a concentration 0.5 units of DNA per microliter
Calibration via the standard given which was a concentration 1.0 units of DNA per microliter |
Calibration via the standard given which was a concentration 2.0 units of DNA per microliter
Calibration via the standard given which was a concentration 5.0 units of DNA per microliter |
Bonus Picture
For the set-up, this picture represents what the set up looked like right before the group closed the flap to take the picture. The camera was set in the cradle and the lens was 6.8 cm away from the drop that was on the superhydrophic side of the glass slide. The box covered the Fluorimeter to protect the SyberGreen Dye from exposure to light. After close the flap, three pictures were taken using the timer on the iPhone to delay the taking of the pictures until after the flap was closed.
Placing Samples onto the Fluorimeter
Ensure that superhydrophic side of the slide is facing up when the slide is placed in the Fluorimeter
Turn On Fluorimeter by flipping the switch on the side of the Fluorimeter
Pipette 80 μL of SYBR Green I dye in between the first two clear circles on the glass slide (light from Fluorimeter should be absorbed by drop if the drop is in the proper place)
Replace the pipette tip and then pipette 80 μL of the PCR sample mix or the calibration sample on top of the SYBR Green I dye droplet
Make sure the light shines through the center of the drop and the light is focused through the opposite side of the drop
Ensure the iPhone rests in the cradle at least 4 cm away from the droplet
Set phone timer for 3 seconds and adjust camera as described in above calibration section
Make sure the iPhone camera focuses on the droplet
Cover the Fluorimeter with the box and press the button to take the picture on the iPhone and quickly close the flap to block all outside sources of light
Take at least three pictures of each sample tested to ensure that a good picture is obtained
Remove the used slide and dispose of properly
Repeat the above setup for each sample
Data Analysis
Representative Images of Negative and Positive Samples
Negative Sample
Positive Sample
Image J Values for All Calibrator Samples
In the calibration section of the chart listed above, picture 1 correlates to H2O, picture 2 correlates to 0.25 units of DNA per microliter, picture 3 correlates to 0.5 units of DNA per microliter, picture 4 correlates to 1.0 units of DNA per microliter, picture 5 correlates to 2.0 units of DNA per microliter, and picture 6 correlates to 5.0 units of DNA per microliter. Picture 6 served as the positive control while picture 1 served as the negative control when checking the results of the PCR experiment.
Calibration curve
PCR Results Summary
Our positive control PCR result was 0.0833 μg/mL
Our negative control PCR result was 0.0833 μg/mL
Observed results
Patient 1 ID# 58575 :
For all images of patient 1, the droplets all did not produce any type of green color when the drop samples were placed on top of the SYBR Green I dye. The drops themselves actually appeared to have a dark center. The concentration of DNA in these samples was the same as the positive and negative controls to which they were compared against. This is because the amount of PCR solution was 100μL which was then pipetted into the 500 μL solution given which resulted in 600 μL. After this solution was created, 80 μL was then pipetted onto the 80 μL of syber green dye which produced and amount which was 0.0833 μg/mL. Based upon the results of patient 1's data, they did not contain the diseased gene sequence.
Patient _____ :
Conclusions
Patient _____ :
Patient _____ :
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is composed of a nitrogenous base, a five carbon sugar, and at least one phosphate group. They are organic molecules that serve as monomers or sub units of nucleic acids, like DNA and RNA. A Polymorphism is a variation in the coding and non coding DNA sequence among a population. It is a discontinuous genetic variation resulting in the occurrence of several different forms or types of individuals among the members of a single species. The SNP analyzed is found in the Homo Sapien species and the chromosome in which it is found is 8:19956018. The clinical significance of the SNP is that is a pathogenic mutation which can cause great harm to the recipient of the mutation. In addition, it is associated with the LPL (lipoprotein lipase) gene and linked to coronary heart disease. A few of the functions of LPL are Apolipoprotein Binding, Heparin Binding, Lipoprotein Lipase Activity, Phospolipase activity, and Protein Binding. A polymorphism can result in something known as an allele which is a variant form of a gene. Some genes - which are located on the exact same spot on a chromosome - have a variety of different forms which are all alleles of that gene. The polymorphism which results in this pathogenic associated allele is AGT. The numerical position of AGT is 19956018. The normal chromosome of a healthy individual not containing this diseased allele is AAT. The mutation of an A to a G results in a diseased gene that is linked with coronary heart disease.
Primer Design and Testing
The diseased primer had no matches because the humane genome does not have the mutation of AGT. Therefore, there would be no match in accordance with the natural human genome tested in the website. While polymorphisms, unique alleles and variations are all what helps to make each individual have a slightly unique genome, the AGT mutation is what can lead to a person having coronary heart disease. The diseased primer has no results while the healthy, non-diseased primer produces a result.