BME100 s2015:Group3 9amL5

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OUR TEAM

Emily Santora
Christina Salas
Steven Mills
Austyn Howard

LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone
    • Flash: Inactive
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest setting
    • Saturation: Highest setting
    • Contrast: Lowest setting


Calibration

  1. Turn on the Blue LED excitation light.
  2. Turn on the smart phone camera and adjust the camera setting as listed above.
  3. Place the smart phone in a cradle of the Fluorimeter so a picture of the side of the drop will be taken.
  4. Adjust the camera to a distance where the first two rows of the slide will be as close as possible for a clear picture of the drop.
  5. Record the distance between the smart phone cradle and the drop using a ruler.
  • Distance between the smart phone cradle and drop = 9.25 cm


Solutions Used for Calibration

Initial 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. Pipette a 80 μL drop of SYBR GREEN I in the middle of the first two rows of the slide, then add 80 μL of the calf thymus solution.
  2. Position the drop by moving the slide so the blue LED light is focused to the middle of the black fiber optic fitting on the other side of the drop.
  3. Set the camera to a 3 second timer to take a picture of the drop.
  4. Take three focused pictures of the drop.
  5. Pipette the 160 μL drop from the surface of the slide and move the slide to the next testing position.
  6. Repeat steps 1-5 for each trial.


Data Analysis

Representative Images of Negative and Positive Samples

With DNA (Positive Signal)

With DNA (positive signa


No DNA

No DNA)


Image J Values for All Calibrator Samples


File:Data tableq.jpeg


Calibration curve
Graph7.jpg


PCR Results Summary

  • Our positive control PCR result was 0.1151655833 μg/mL
  • Our negative control PCR result was 0.114970375 μg/mL

Observed results

  • Patient 27468 : The drop was blue, and no green color appeared in the picture. The average value of the initial PCR product concentration for patient one was -0.01763836111 μg/mL.
  • Patient 35294 : The drop was blue, and no green color appeared in the picture. The average value of the initial PCR product concentration for patient one was -0.07353509722 μg/mL.

Conclusions

  • Patient 27468 : Inconclusive, because the first trial showed a positive result, while the remaining trials showed a negative result.
  • Patient 35294 : Negative, because all of the trials showed a negative result.




SNP Information & Primer Design

Background: About the Disease SNP

SNP is a DNA sequence associated with disease. Mostly, SNPs occur within two alleles. SNP density can fluctuate depending on rate of mutation and genomic recombination. Furthermore, when a single nucleotide mutation occurs in a biological species, a SNP will occur. Also, SNPs mostly occur within non-coding regions rather than coding regions.

Primer Design and Testing

The non-disease primer (shown below) yielded results, because it did not include the SNPs. Picture 1eks.jpg

The disease primer (shown below) did not yield results, because it included the SNPs. Picture 2eks.jpg