BME100 s2015:Group18 12pmL5

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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: Jenna Taras
Name: Eyerusalem
Name: Walter C. Bregon
Role(s): Chief of Neurosurgery
Name: Nathan LeFort
Name: Abdurrahman Darwish
Name: Hau Nguyen


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S5
    • Flash: Inactivated
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest setting available
    • Saturation: Highest setting available
    • Contrast: Lowest setting available

Even though we set these parameters, the Galaxy was sensitive enough to get good calibration results with DNA concentration.

Calibration
1. Turn on Blue LED excitation light.
2. Turn on the camera of the smartphone and adjust the camera setting as those listed above. If your phone won't allow you to change settings than don't worry about this. Just hope that you bought a good enough phone that has high quality picture taking capabilities.
3. Place the phone on the cradle as close to a 90 deg angle as possible from the slide so that it takes a picture of the SYBR GREEN/Calf Thymus DNA solution drop sideways. You may need to raise the fluorimeter using the plastic trays provided in order to make sure the camera is sideways to the drop. This will allow ImageJ to filter the blue light easier.
4. Adjust the distance between the smartphone on its cradle and the first two rows of the slide (where the drop will be placed) so that it is as close as humanly possible without causing the image to blur. This distance is atleast 4cm away from the drop and should be measured with a metal ruler.
5. Record this distance and try not to move the camera, cradle, or fluorimeter because this can change the image due to the difference in light collect between the different lengths.

  • Distance between the smart phone cradle and drop "picture distance" = 7.3 cm


Solutions Used for Calibration

Initial [2X Calf Thymus DNA Soln] (ug/ml) Vol of the 2X DNA Soln (uL) Vol of the SYBR GREEN I Dye soln (uL) Final [DNA] in SYBR GREEN I soln (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


Photo of fluorimeter set-up


IMG 7300.jpeg


Placing Samples onto the Fluorimeter

  1. Deposit a 80 uL drop of SYBR GREEN I in middle of the 1st 2 rows of the slide using the pipettor so the drop is stationary & round.
  2. Add 80 uLs of the 2X DNA soln.
  3. Align the drop by moving the slide so that the blue LED light goes through the middle of the drop and is not refracted to any side.
  4. Set up the timer on your camera (of your smartphone) to take a picture of the drop after you have closed the light (black) box over the fluorimeter & camera. This box removes as much light as possible so that the SYBR GREEN doesn't start to react before we need it to causing the results to be imprecise.
  5. Take 3 images of the drop while keeping the camera focused and not moving anything.
  6. Remove the box while not moving the phone. If you need to adjust for any accidental movement of the cradle, the phone, or the fluorimeter setup at any time, be sure to use the metal ruler to set the same "picture distance" you used before.
  7. Use the pipettor to remove the 160 uL drop from the surface.
  8. Move the slide to the next position so that the next sample drop will have a clean surface to 'sit' on.
  9. Repeat steps 1-8 for all the other concentrations of Calf Thymus DNA as outlined on the table above.
  10. You can use another slide if you make a complete mess of a slide. I paid for them so you don't have to worry about expense.
  11. Don't forget to do the zero [DNA] solution.


Data Analysis

Representative Images of Negative and Positive Samples

Sample Droplet with no DNA

‎


Sample Droplet with DNA (positive signal

Bme100 lab5 image2.jpg


Image J Values for All Calibrator Samples

Table 1: Edited Raw Data

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 74904 128.083 9593914 45481 9548433
1 61875 123.303 7620386 91250 7529136
0.5 52292 87.918 4597419 114789 4482630
0.25 43266 59.326 2566780 120847 2445933
0.125 25922 32.018 829979 56835 773144
0 36072 71.246 2570001 239501 2330500





Calibration curve
Bme200 lab5 calibrationcurve.PNG


PCR Results Summary

  • Our positive control PCR result was 0.6510 μg/mL
  • Our negative control PCR result was -0.3926 μg/mL

Observed results

  • Patient 41180 : The image of the drop was green. The 1st, 2nd and 3rd replicates, respectively were: -0.4362, -0.5876, 0.7658
  • Patient 68784: The image of the drop was almost no green to clear in color. The 1st, 2nd and 3rd replicates, respectively were: -0.5428, -0.3680, -0.3623

Conclusions

  • Patient 41180 : Patient 1 had inconclusive results possibly due to sample contamination or possibly because of forgetting to put the cyber green in to the sample droplet or allowing the droplet to be exposed to light for longer than it should have, etc. Patient 1 probably was negative for the [DNA] with respect to cyber green in PCR but it turned out to be inconclusive because two of the replicates were negative but one was positive.
  • Patient 68784 : Patient 2 was negative because all three replicates were negative just like the negative sample. This means that the sample droplet showed very little green if any to clear in color. Again the causes for this are the same as previously written for the patient 1.




SNP Information & Primer Design

Background: About the Disease SNP

SNP stands for "single nucleotide polymorphism". Polymorphisms at specific points in the DNA sequence are what make each individual different from the next. They are what leads to genetic variation between people. SNPs are heavily influential to how an individual looks, acts, if they are susceptible to certain diseases, and even drug efficacy and side-effects.

In this lab, the disease was coronary heart disease in humans. Students looked at the gene SNP rs268 to see if the disease was present. The gene that codes for the disease was present if the polymorphism changed from AAT to AGT, which is the disease-associated allele.

Primer Design and Testing


Non diseased Forward primer (20): 5’-AATCTGGTCTATGAGATCAA

Non diseased Reverse primer (20): 5’-GAAACACCAGGGCTCAGGGT

Diseased Forward primer (20): 5’-AATCTGGGCTATGAGATCAG

Diseased Reverse primer (20): 5’-GAAACACCAGGGCTCAGGGT

In a Non diseased person, when you run the PCR, the reverse prime will duplicate, however, the forward primer will not. Therefore, you are only getting the result for half of the strand. Additionally, the reverse primer will not pair up with the forward primer. In a diseased primer, both forward and reverse primers duplicate, and they will base pair according to their nucleotide sequence.

A will pair with T

C will pair with G