BME100 f2014:Group11 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Aliya Yano
Name: Breanna Corrigan
Name: Julian Lopez
Name: Carlos Cabanes
Name: Mohammed Almaimani


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone5S
    • Flash: No Flash
    • ISO setting:800
    • White Balance: Auto
    • Exposure:Auto
    • Saturation:Auto
    • Contrast:Auto
    • Set phone to video mode then simply took three screenshots of to get our pictures


Calibration

  • Distance between the smart phone cradle and drop = 5cm


Solutions Used for Calibration

Initial Concentration of n2X 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. Choose a camera to use, set it to the specifications described in the workbook and place it in the stand.
  2. Set up the Flourimeter.
  3. Place slide in the flourimeter with the smooth side down and the rough side up.
  4. Place one, 80 micro-mL drop of SYBR Green on the slide, between two of the dots on the slide.
  5. Place one, 80 micro-mL drop of a calf thalmus solution on the slide so it combines with the SYBR Green.
  6. Adjust the slide so the light shines through the drops.
  7. Adjust and record camera distance.
  8. Start camera video and close the fluorimeter.
  9. Wait a few seconds and remove camera, take screenshots on the video at different times and send them to be analyzed.
  10. Discard the droplets and slide.
  11. Repeat.


Data Analysis

Representative Images of Negative and Positive Samples


Positive
Positive Sample


Negative
Negative Sample


Image J Values for All Calibrator Samples

Final DNA Concentration in SYBR Green I Solution (µg/mL) Area Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the Background RAWINTDEN DROP - BACKGROUND
2.5 72592 27.453 1992874 0 1992874
2.5 81840 24.854 2034060 6 2034054
2.5 69124 35.062 2423628 0 2423628
1 100748 32.737 3298170 69 3298101
1 90320 36.084 3259073 6 3259067
1 90392 36.403 3290496 67 3290429
0.5 3578 18.658 66760 1605 65155
0.5 52088 15.451 804792 1540 803252
0.5 62452 13.06 815628 2564 813064
0.25 113076 16.741 1892954 9935 1883019
0.25 68308 26.692 1823278 9604 1813674
0.25 60128 29.933 1799822 8492 1791330
0.125 71016 27.528 1954937 13177 1941760
0.125 52480 35.86 1881914 3204 1878710
0.125 73144 73144 27.193 4377 -4349.807
0 67164 28.526 1915935 4863 1911072
0 78472 21.653 1699177 3126 1696051
0 73296 22.284 1633341 1343 1631998



Final DNA Concentration in SYBR Green I Solution (µg/mL) 1 2 3 Average Standard Deviation
2.5 3298101 3259067 3290429 3282532.333 20680.45689
1 1992874 2034054 2423628 2150185.333 237701.7397
0.5 1883019 1813674 1791330 1829341 47810.13854
0.25 1911072 1696051 1631998 1746373.667 146184.3099
0.125 1941760 1878710 4349.807 1274939.936 1100814.826
0 65155 803252 813064 560490.3333 429001.0352


Calibration curve

calibration
Image J Values for DNA Samples

PCR Product Tube Label Area Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the Background RAWINTDEN DROP - BACKGROUND
P 66936 28.847 193087 3483 189604
P 47080 37.904 1784535 3396 1781139
P 58340 32.315 1885230 8297 1876933
N 36472 34.757 1267652 0 1267652
N 59360 22.418 1330703 0 1330703
N 48700 27.63 1345571 16 1345555
1.1 53964 25.715 1387673 16 1387657
1.1 52332 35.469 1856168 6575 1849593
1.1 56356 32.198 1814544 1574 1812970
1.2 39616 34.895 1382389 0 1382389
1.2 51536 27.49 1416732 0 1416732
1.2 69756 24.391 1457486 0 1457486
1.3 50404 36.576 1843555 273 1843282
1.3 50656 37.629 1906116 6616 1899500
1.3 6287 26.116 164194 1234 162960
2.1 65224 27.275 1778976 70 1778906
2.1 43356 38.869 1685201 2480 1682721
2.1 4526 31.611 143071 212 142859
2.2 65880 37.459 2467789 3149 2464640
2.2 65064 35.556 2313390 683 2312707
2.2 5838 35.754 208730 837 207893
2.3 37080 37.087 1375187 2508 1372679
2.3 42904 33.525 1438374 3256 1435118
2.3 36256 36.624 1327825 400 1327425



PCR Product Tube Label 1 2 3 Average Standard Deviation
P 189604 1781139 1876933 1282558.667 947737.5956
N 1267652 1330703 1345555 1314636.667 41361.99527
1.1 1387657 1849593 1812970 1683406.667 256780.4705
1.2 1382389 1416732 1457486 1418869 37594.08104
1.3 1843282 1899500 162960 1301914 805689.0535
2.1 1778906 1682721 142859 1201495.333 918066.4754
2.2 2464640 2312707 207893 1661746.667 1261363.854
2.3 1372679 1435118 1327425 1378407.333 54074.54035



Calculating Concentration of PCR Product

PCR Product Tube Label MEAN RAWINTDEN DROP - BACKGROUND PCR PRODUCT CONCETRATION INITIAL PCR PRODUCT CONCENTRATION
P 1282558.667 0.313086477 3.75703772
N 1314636.667 0.348630203 4.183562439
1.1 1683406.667 0.757242338 9.086908057
1.2 1418869 0.464123861 5.569486335
1.3 1301914 0.334532972 4.014395664
2.1 1201495.333 0.223265012 2.679180139
2.2 1661746.667 0.733242179 8.798906142
2.3 1378407.333 0.419290691 5.031488298

PCR Results Summary

  • Our positive control PCR result was 3.757 μg/mL
  • Our negative control PCR result was 4.183 μg/mL

Observed results

  • Patient 81166 : All images taken of patient one's DNA samples seemed to be negative. None of them glowed green. All of this patients concentration results were above the negative control.
  • Patient 54283 : All images taken of patient two' DNA samples appeared to be negative as well as none of them glowed green like the positive sample did. However, not all of the values of concentration found for patient two were above the negative control.

Conclusions

  • Patient 81166 : Based on the images alone, this patient tested negative.The data calculated shows all concentrations to be above both the negative and positive controls which suggests the sample to be negative as the negative concentration was found to be higher than the positive. The final conclusion is this patient is negative.
  • Patient 54283 : Based on the images alone, this patient tested negative. The data calculated shows two of the three concentrations to be above both the negative and positive control and one below. This still impliess that this patient is negative.

Examining only the data collected, it is hard to determine a result as all the data found using Image J is relatively similar. The positive and negative control concentrations were very near to each other with the negative control having a higher concentration than the positive control. The data collected for the patients ranged both above and below the controls.This suggests an error was made and further testing would be needed to get a definitive result.




SNP Information & Primer Design

Background: About the Disease SNP A nucleotide is a subunit of a nucleic acid and a polymorphism is when two or more clearly different phenotypes exist within the same population. Therefore, a SNP, or single nucleotide polymorphism, is simply a DNA sequence variation where a single nucleotide is different when compared to others of the population. The variation rs1699154 is found in Homo Sapiens a.k.a humans. It is located on the chromosome 21:34370656. This SNP is pathogenic and associated with the KCNE2 gene-- which stands for potassium voltage-gated channel-- and the cardiac disease Long QT Syndrome.

Primer Design and Testing The KCNE2 gene stands for potassium voltage-gated channel Isk-related family, member 2. The non-disease allele is TTC but the disease-associated allele contains the sequence is CTC. The numerical location of this SNP is 34370656. The numerical position needed for the non-disease reverse primer is exactly 200 bases to the right of the SNP at 34370856. the non-disease forward primer is 5'-catggtgatgattggaatgt, the reverse primer sequence is 5'-cccttatcagggggacattt. Similarly, the disease forward primer is 5'-catggtgatgattggaatgc and the disease reverse primer is cccttatcagggggacattt.

calibration calibration