BME100 f2014:Group1 L5

BME 100 Fall 2014	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: Hawley Helmbrecht Name: Sarah Fakhoury Role(s) Name: Prerna Gupta Role(s) Name: Jonathan Riecker Name: Michael Pineda Role(s) Name: Timothy Black LAB 5 WRITE-UP Procedure Smart Phone Camera Settings Type of Smartphone: Samsung S3 Flash: No Flash ISO setting: 800 White Balance: Auto Exposure: + 2.0 Saturation: No setting to affect Saturation Contrast: Auto Contrast Off Setup for Smart Phone Fluorimeter Calibration Place the phone in the cradle with the camera lens lined up so that it shows a view of the slide from the side (edge-on). Once the camera is set up in the cradle and lined up with the side of the slide, adjust the slide so that the light illuminates the center of the drop and the drop focuses light on the other side of it. After adjusting the slide itself, change the distance of the smartphone from the fluorimeter in order to keep the drop in focus but keep the view close. Distance between the smart phone cradle and drop = 5 cm Solutions Used for Calibration Concentration of 2X Calf Thymus DNA solution(micrograms/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Dye Solution (μL) Final DNA concentration in SYBR Green I solution (μg/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 0 Placing Samples onto the Fluorimeter Find the rough side of the slide (making sure to only touch the slide while wearing gloves) Place the slide, with the rough side up, in the fluorimeter Locate the two clear circles in the middle of the slide and add 80 μL of SYBR Green I solution On the same circle with the SYBR Green I solution, place 80 μL of Sample/Calibration drops Adjust the slide so that the middle of the drop is illuminated by the light and the drop becomes focused on the side Data Analysis Representative Images of Negative and Positive Samples Positive Control: Negative Control: Image J Values for All Calibrator Samples Solutions Used for Products PCR Product Label Tube Volume of the DILUTED PCR Product Solution (μL) Volume of the SYBR GREEN I Dye Solution (μL) Dilution 1 Dilution 2 Total Dilution (Simplified Fraction) Positive 80 80 1/6 1/2 1/12 Negative 80 80 1/6 1/2 1/12 G1 1-1 80 80 1/6 1/2 1/12 G1 1-2 80 80 1/6 1/2 1/12 G1 1-3 80 80 1/6 1/2 1/12 G1 2-1 80 80 1/6 1/2 1/12 G1 2-2 80 80 1/6 1/2 1/12 G1 2-3 80 80 1/6 1/2 1/12 Calibration curve PCR Results Summary PCR Product Label Tube Mean Rawintden Drop-Background PCR Product Concentration(μg/mL) Initial PCR Product Concentration(ug/mL) Positive 5305678 1.19 .0992 Negative 1928602 .23 .0192 G1 1-1 5306397 1.19 .0992 G1 1-2 6833849 1.63 .1358 G1 1-3 5792721 1.33 .1108 G1 2-1 1287212 .048 .0040 G1 2-2 3698137 .735 .0613 G1 2-3 1883281 .218 .0182 Our positive control PCR result was 1.19 μg/mL Our negative control PCR result was .23 μg/mL Observed results Patient 43236 : The concentration of this sample was 1.38ug/mL. The drop of sample when put into the SYBR I turned a bright green and was easily visible by the naked eye. Patient 55773 : The concentration of this sample was .33ug/mL. The drop of sample when put into the SYBR I stayed fairly clear and did not glow as green. Conclusions Patient 43236 :This patient was very close to the positive control. This would mean that this patient does have the disease because the genetic sequence in question amplified to the same extent as the positive control. Patient 55773 :This patient was very close to the negative control. This leads us to believe that the patient does not have the disease because the genetic sequence in question did not amplify as much and instead only created as much PCR product as the negative control. SNP Information & Primer Design Background: About the Disease SNP A SNP stands for a Single Nucleotide Polymorphism. It is a very natural occurrence in the human genome. SNPs occur as sequence variations, fixed at different locations on the genome. SNPs can result in variations in the phenotype characteristics of the individual, such as hair type or their physical structure. They also affect the person's immunity and their reaction to certain medicines. These polymorphisms or genetic variations do more good than harm as they help in determining genetic linkage as well as drug efficacy and disease risk. SNPs are a collection of polymorphisms, and can occur as single nucleotide substitutions. Nucleotides are the essential building blocks of DNA and RNA. Changes or substitutions of these nucleotides have a direct effect on the individual. In general, SNPs are a major factor that contributes to biological diversity among organisms, and can be categorized into specific types, all with various effects on the human genome. Primer Design and Testing The results of our primer test were as follows: The non-disease primer was successful while the disease primer test did not yield any results. This is due to the single nucleotide polymorphism in the disease primers. Match Non-disease Primers: No Match Disease Primers: