BME100 f2014:Group2 L5

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Contents

OUR TEAM

Victoria Bowman
Victoria Bowman
Andre Dang
Andre Dang
Chandler Heaton
Chandler Heaton
Jordan Shinn
Jordan Shinn
Abigail Weiss
Abigail Weiss
Image:AZPP.jpg
Alena Zapata


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 4
    • Flash: Off
    • ISO setting: Default
    • White Balance: Default
    • Exposure: Default
    • Saturation: Default
    • Contrast: Default


Calibration

[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]

  • Place the camera in the cradle and set it 4cm away from the fluorimeter. Place plates under the fluorimeter so that when looking through the camera the slide is at eye level and the back of the slide can not be seen.


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL) Volume of the 2X DNA Solution (μL) Volume of 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. Step one, Turn the fluorimeter light on and pull the slide so that the light shines in the middle of two dots on the slide
  2. Step two, Get a new tip on the micro pipette and place 80mL of the SYBR Green I on the slide in between two dots on the slide. Dispose of this tip in the plastic cup .
  3. Step three, Replace micro pipette with a new tip and place 80mL of the DNA sample onto the SYBR Green I dot
  4. Step four, Set camera timer and close the box so that no light can enter when the timer goes off and the picture is taken. Take three pictures this way.
  5. Step five, Remove the sample from the slide using the micro pipette and dispose of it in a plastic cup along with the tip
  6. Step six, Push slide so that the next row of dots is in the lights pathway and complete steps two through five using your next desired DNA sample


Data Analysis

Representative Images of Negative and Positive Samples
Positive Control Negative Control


Image J Values for All Calibrator Samples

INTDEN Values

Mean and Standard Deviation

PCR Dilution


Calibration curve


PCR Results Summary

  • Our positive control PCR result was ____ μg/mL
  • Our negative control PCR result was ____ μg/mL

Observed results

  • Patient 13209:

1 2 3

  • Patient 26070:

1 2 3

Conclusions

  • Patient 13209: Results suggest that patient 13209 tested closer to the positive value.
  • Patient 26070: Results suggest that patient 26070 tested closer to the negative value.




SNP Information & Primer Design

Background: About the Disease SNP

SNP is a variation associated with the gene KCNE2. KCNE2 regulates neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. This SNP variation is located on chromosome 21:34370656 . This SNP variation is significant because it is pathogenic and linked to the disease congenital long QT syndrome. This disease can cause heart rhythm disorders.

Primer Design and Testing


We found the numerical position of the SNP to be 34370656. From this position we found the non-disease forward primer to be 5’-CATGGTGATGATTGGAATGT. From the position 200 bases to the right of the SNP we receive our non-disease reverse primer which is 5’-CCCTTATCAGGGGGACATTT. From this information we were able to determine our disease forward primer to be 5’-CATGGTGATGATTGGAATGC and our disease reverse primer to be 5’-GAACACATTATTGGCTTTAA. When we tested for the non-disease reverse and forward primers using the In-Silico PCR we got a result that matched to chr21:35742936+35743155. When we tested for the disease reverse and forward primers we got no result.

Results for Non-Disease Forward and Reverse Primers

Image:Pcr.png


Results for Disease Forward and Reverse Primers

Image:Pcr2.png




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