BME100 f2014:Group20 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

Karthik Nambiar
Aleks Radchenko
Bjorn Houman
Ryan Giudice
Jeremy Ellis
Connor Seiferth


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5s
    • Flash: No Flash
    • ISO setting:N/A
    • White Balance: N/A
    • Exposure:N/A
    • Saturation:N/A
    • Contrast:N/A


Calibration
The camera was placed in the cradle at a right angle, and the height was adjusted so that the camera was able to get a straight shot on the picture. The distance from camera to slide was carefully measured, and the cradle was placed inside the lightbox properly.

  • Distance between the smart phone cradle and drop = 11 cm


Solutions Used for Calibration

Amount of Calf Thymus DNA solution(micrograms/mL) Volume of the 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
.5 80 80 0.25
0.25 80 80 0.125
0 80 80 0



Placing Samples onto the Fluorimeter

  1. Place the slide on the Flourimeter with the rough side facing up
  2. Place 80 micro-liters of SYBR green solution on the slide on the two circles in the middle
  3. Following that, place an additional 80 micro-liters of the calibration solution in with the SYBR green solution
  4. Then, light the drop with the blue light from the Flourimeter by adjusting the position of the slide
  5. Using the phone camera, take a covered picture of the sample
  6. Using the ImageJ software, collect the data
  7. Repeat each of the previous steps for each subsequent sample


Data Analysis

Representative Images of Negative and Positive Samples This doesn't have DNA: NoDNAG202014.jpg This has DNA: DNAG202014.jpg


Image J Values for All Calibrator Samples


G2023(Mg/mL) Area Mean RawIntDen Drop RawIntDenBackground Drop-Background
0 112704 37.029 4173330 4173330 80486
0.125 115052 115.304 132565969 13265969 355717
0.25 76220 42.315 3225427 3225247 211612
0.5 108269 84.101 9105547 9105547 237419
1 99946 125.025 12475765 12475765 46683
2.5 104836 183.136 19199198 19199198 108537
0.125 104976 114.33 12001882 12001882 509621
0.25 74493 40.904 3042076 3042076 65389
0.5 112648 81.69 9202174 9202174 240454
1 90873 126.962 11537375 11537375 53851
2.5 106250 177.936 18905736 18905736 107969
0.125 117716 145.994 17185841 17185841 17037984
0.25 72426 39.731 2877591 2877591 48997
0.5 113313 81.15 9195343 9195343 241627
1 98512 126.812 12492486 12492486 58334
2.5 105285 168.462 17736545 17736545 91600
0 116198 36.957 4294334 4294334 285141
0 109924 35.248 3874568 3874568 76169


Calibration curve

BME100Lab51.png

PCR Results Summary

  • Our positive control PCR result was .34 μg/mL
  • Our negative control PCR result was 0 μg/mL

Observed results

  • Patient 58272: .35 μg/mL
  • Patient 80239 : .68 μg/mL

Conclusions

  • Patient 58272 : This patient seems to be negative, despite values that are awfully close to the positive control, they are significantly lower than the 2nd patient, which implies a negative outcome.
  • Patient 80239 :This patient seems to be positive, as the estimated values are far above what is to be expected of a positive sample




SNP Information & Primer Design

Background: About the Disease SNP

SNP stands for Single-nucleotide polymorphism; it is a disease in which a single nucleotide in a DNA sequence differs from that of the other chromosome in the pair or from other individuals of the species. There are 4 nucleotides used in a DNA sequence: two purines called adenine and guanine and two pyrimidines called cytosine and thymine. In a SNP, adenine may be changed out for a guanine to create a singe-nucleotide change in the DNA code. There are 10 million SNPs in the human genome where a single SNP occurs within roughly every 300 nucleotides. Most SNPs will have no affect on an organism in its environment but certain SNPs can have outstanding effects. Some of there effects include resistance to certain drugs and toxins. Scientists often use SNPs as "biological markers" where they use the singe alternate nucleotide as an indicator for the location of a certain gene. They can also be used to track genes and determine if a given gene is inherited by the offspring of two individuals.

Primer Design and Testing

The results of our primer test were conclusive. Each trial ran produced the expected results, our positive control gave off bright green light and our negative control along with the water trial was clear. As we ran the two different patient samples three times each we observed that both were consistent to themselves, meaning our results were not only accurate but repeatable as well.