BME100 f2014:Group4 L5

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OUR TEAM

Name: Anyssa Iwamoto
Name: Shreya Ramkumar
Name: Izzy Ortiz
Name: Larrison Black
Name: Zach Sledge
Name: Dominique Stewart


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5C
    • Flash: Off
    • ISO setting: N/A
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration

  • Distance between the smart phone cradle and drop = 7.8 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL) Volume of the 2X DNA Solution (uL) Volume of the SYBR Green I Solution (uL) Final DNA Concentration in SYBR Green I Solution (ug/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. Slide the glass slide onto the fluorimeter with the smooth side down and adjust the glass so that the blue light is shining in between two white circles.
  2. Set up the phone camera to the three second timer and turn the flash off. (If the flash is not able to be disabled, skip this step)
  3. Place the phone in the holder and make sure the glass slide is level with the camera and in focus.
  4. Measure the distance between the phone and the fluorimeter (Make sure the camera is more than 4 cm away, in our experiment we used 7.8cm)
  5. Place 80 uL drops of SYBR Green I solution in the between the two circles that the blue light is illuminating. (Remember to use a new micropipette tip for each different solution and discarding the tips in the red disposable cup.)
  6. Place 80 uL drops of the sample solution onto the SYBR Green I drop that is already on the glass slide.
  7. Place the box over the fluorimeter with the opening on the side of the camera.
  8. Focus the image and click on the timer before covering the box with the lid OR cover the box as much as possible while clicking the shutter manually.
  9. Using the micropipette, remove all 160 uL solution from the slide and discard the solution into the red disposable cup.
  10. Move the slide so the blue light shines between two new white circles.
  11. Repeat this procedure for each sample three times.

Put the glass slide in the sharps container once all the possible white circle have been used.

Bonus: Images of Setup

Desc

Above is an image of the basic setup without the black box over it. Below is an image of the setup with the black box over it but without closing the final side so that we could take the picture.

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Data Analysis

Representative Images of Negative and Positive Samples

Negative Sample Desc Positive Sample Desc


Image J Values for All Calibrator Samples

PCR Product Label Tube Area Mean Pixel Value RAWINTDEN of Drop RAWINTDEN of Background RAWINTEDEN (Drop-Background)
5 498 238.514 118780 755 118025
5 498 237.96 118504 1135 117369
5 498 253.303 126145 1918 124227
2 498 242.853 120941 750 120191
2 498 201.57 100382 659 99723
1 498 170.665 84991 687 84304
1 498 163.55 81448 1206 80242
1 498 171.643 85478 786 84692
0.5 498 115.305 57422 740 56682
0.5 500 113.478 56739 840 55899
0.5 484 113.291 54833 799 54034
0.25 484 68.285 33050 632 32418
0.25 484 76.911 34321 722 33599
0.25 484 70.155 33955 1465 32490
0 484 7.395 3579 982 2597
0 484 7.795 3773 693 3080
0 484 7.905 3826 565 3261

Image J Data

PCR Product Label Tube RAWINTEDEN (Drop-Background) AVERAGE Standard Deviation
2.5 119873.67 3784.34
1 109957.00 14473.06
0.5 83079.33 2464.85
0.25 55538.33 1360.34
0.125 32835.67 662.05
0 2979.33 343.26

Calibration curve
Desc


The last two values plateau in a non-linear way with the graph above throwing off the line of best fit which is why we excluded them on the graph below.


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PCR Results Summary

PCR Product Label Tube Average PCR Production Concentration Initial PCR Production Concentration
G1-1 3964 -0.0349 -0.0029
G1-2 2602.5 -0.0436 -0.0036
G1-3 1945.5 -0.0478 -0.0040
G2-3 5176 -0.0271 -0.0023
G2-2 4825 -0.0294 -0.0024
G2-3 3850 -0.0356 -0.0030
Negative 82357.5 0.4666 0.0389
Positive 122055 0.7206 0.0600
  • Our positive control PCR result was 0.0600 μg/mL
  • Our negative control PCR result was 0.0389 μg/mL

Observed results

  • Patient 85470 : The values on this patient were -.0029, -.0036, and .0040 μg/mL. The drops were not green at all so it was negative.
  • Patient 90113 : The values on this patient were -.0023, -.0024, and .0030 μg/mL. The drops were not green at all so it was also negative.

Conclusions

  • Patient 85470 : The results suggest that Patient 85470 is closer to the negative value.
  • Patient 90113 : The results suggest that Patient 90113 is closer to the negative value.


SNP Information & Primer Design

Background: About the Disease SNP
SNP is the abbreviation meaning Single Nucleotide Polymorphism and affect appearance, respond to drugs, and pre-disposition to diseases. In this lab, the particular SNP that is being analyzed is rs16991654, which is called Homo Sapiens (humans) in latin. On the National Center for Biotechnology (NCBI) page, numerous information on this SNP's can be found including the clinical significance, which is pathogenic or the disease linked to this SNP, which is Long QT syndrome (LQTS). The area of information that we focused on for the majority of the time is the gene sequence, which is KCNE2 where we analyzed the numerical position of the SNP (34370656) and had to not the non-disease forward primer, non-disease reverse primer, the disease forward primer, and the disease reverse primer. KCNE2 is gene coding which regulates numerous functions such as the neurotransmitter release, the heart rate, insulin secretion, neuronal excitability, and several others.

Latin Name Homo Sapien (Humans)
Chromosome 21:34370656
Clinical Significance Pathognic
Associated Gene KCNE2 (Potassium Voltage-Gated Chanel, Isk-related family member)
Associated Disease Long QT Syndrome (LQTS)
Numerical Postion 34370656
Numerical Position (200 bases to the right) 34370856
Non-Disease Forward Primer CAT-GGT-GAT-GAT-TGG-AAT-GT
Non-Disease Reverse Primer CCC-TTA-TCA-GGG-GGA-CAT-TT
Disease Forward Primer CAT-GGT-GAT-GAT-TGG-AAT-GA
Disease Reverse Primer CCC-TTA-TCA-GGG-GGA-CAT-TT


Primer Design and Testing

The image below shows the UCSC In-Silico PCR website results for the non-disease forward primer and non-disease reverse primer. C1.png

The image below shows the UCSC In-Silico PCR website results for the disease specific primers. The results show that there is "no matches." C2.png