BME100 s2015:Group2 12pmL5
 BME 100 Spring 2015
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Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||
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OUR TEAM
LAB 5 WRITE-UPProcedureSmart Phone Camera Settings
Error creating thumbnail: Unable to save thumbnail to destination 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
Data AnalysisRepresentative Images of Negative and Positive Samples Negative Sample Error creating thumbnail: Unable to save thumbnail to destination Positive Sample Error creating thumbnail: Unable to save thumbnail to destination
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.
Observed results
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 SYBR green dye which produced and amount which was 0.0833 μg/mL. These droplets all did not glow with SYBR Green I dye meaning they looked similar to the negative control which did not glow either.
For all the images of Patient 2, the droplets did not produce any green glow. In fact, the droplets appeared to actually have a dark center when the light was concentrated on them. The results of Patient 2 essentially mirrored those of Patient 1. 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 SYBR green dye which produced and amount which was 0.0833 μg/mL. These droplets all did not glow with SYBR Green I dye meaning they looked similar to the negative control which did not glow either.
The results of Patient 1 were comparable to the results of the negative control. This is due to the fact that neither the negative control nor Patient 1 produced a green glow when paired with SYBR Green I dye on the Fluorimeter. Both samples had the same concentration (0.0833 μg/mL) meaning that their lack of glowing was not due to a discrepancy in the contents, but rather due to the fact that both these samples contained no DNA or negligible amounts of DNA. Because SYBR green I dye glows green in the presence of DNA, Patient 1's genes were not replicated in PCR. The droplet did not glow green because there was no DNA - or a negligible amount of DNA - to cause it to glow. This means that the PCR did not replicate Patient 1's DNA. The most probable cause of this is due to the presence of the diseased gene. The PCR mix contained the healthy primer which would only bind with healthy, non mutated DNA. The primer cannot bind to DNA that contains a gene mutation because the base pairs do not properly match. This means that Patient 1's lack of glowing in the presence of SYBR green I dye can be accredited to the fact there is a negligible amount of DNA (or no DNA) present. This negligible amount of DNA is due to the fact that Patient 1's DNA could not replicate in the PCR reaction because the genes were diseased and would not match with the primer. All this goes to say that Patient 1 contains the diseased gene.
Based upon the lack of glowing green in Patient 2, these results were comparable to the negative control. The negative control was the sample which did not glow. The concentrations of both of these samples was the same as well (0.0833 μg/mL), so the problem was not in a discrepancy of contents. Because SYBR green I dye glows green in the presence of DNA, Patient 2's genes were not replicated in PCR. The droplet did not glow green because there was no DNA - or a negligible amount of DNA - to cause it to glow. This means that the PCR did not replicate Patient 2's DNA. The most probable cause of this is due to the presence of the diseased gene. The PCR mix contained the healthy primer which would only bind with healthy, non mutated DNA. The primer cannot bind to DNA that contains a gene mutation because the base pairs do not properly match. This means that Patient 2's lack of glowing in the presence of SYBR green I dye can be accredited to the fact there is a negligible amount of DNA (or no DNA) present. This negligible amount of DNA is due to the fact that Patient 2's DNA could not replicate in the PCR reaction because the genes were diseased and would not match with the primer. All this goes to say that Patient 2 contains the diseased gene because it did not glow in the presence of the SYBR Green I dye meaning that the PCR could not replicate the diseased genes.
SNP Information & Primer DesignBackground: About the Disease SNP
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. Diseased Primer <a href="http://imgur.com/ruzkEYS"><img src="http://i.imgur.com/ruzkEYS.png" title="source: imgur.com" /></a> NON-Diseased Primer <a href="http://imgur.com/TAV9kPT"><img src="http://i.imgur.com/TAV9kPT.png" title="source: imgur.com" /></a>
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