BME100 s2015:Group2 12pmL5

BME 100 Spring 2015	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
OUR TEAM Name: Levi Riley Name: Alexandria Clark Name: Blossom Mendonca Name: Alina Kilic Name: Trevor Douglass Name: Andre Dang LAB 5 WRITE-UP Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 6 Flash: Off ISO setting: Auto White Balance: Auto Exposure: Auto Saturation: Auto Contrast: Auto Calibration 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 _____ : if DNA is present, SYBR green will react with it and it will show green. DNA can't replicate in PCR if it is diseased because the primers in the PCR won't match will the diseased DNA. If a primer is diseased, then it won't replicate, so very little DNA is present meaning the SYBR Green will not fluoresce because there is very little to no DNA present. 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. 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>