BME100 f2014:Group1 L5

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Contents

OUR TEAM

Name: Hawley Helmbrecht
Name: Hawley Helmbrecht
Name: Sarah FakhouryRole(s)
Name: Sarah Fakhoury
Role(s)
Name: Prerna GuptaRole(s)
Name: Prerna Gupta
Role(s)
Name: Jonathan Riecker
Name: Jonathan Riecker
Name: Michael PinedaRole(s)
Name: Michael Pineda
Role(s)
Name: Timothy Black
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

Description of image


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

  1. Find the rough side of the slide (making sure to only touch the slide while wearing gloves)
  2. Place the slide, with the rough side up, in the fluorimeter
  3. Locate the two clear circles in the middle of the slide and add 80 μL of SYBR Green I solution
  4. On the same circle with the SYBR Green I solution, place 80 μL of Sample/Calibration drops
  5. 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: Description of image

Negative Control: Description of image


Image J Values for All Calibrator Samples

Description of image

Description of image

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 1Dilution 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
Description of image

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: Description of image

No Match Disease Primers: Description of image

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