BME100 s2015:Group7 12pmL5

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OUR TEAM

Name: Alex Kratz
Name: Stephen Geraci
Name: Keawepono Wong
Name: Marvin Vergara
Name: Jose Rodriguez
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LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Galaxy S5
    • Flash:Off
    • ISO setting:Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation:Auto
    • Contrast:Auto


Calibration

  • Distance between the smart phone cradle and drop = 9 cm (Should at least be 4 cm)
  • The camera should be set with a timer of about 3-5 seconds in order to close the box prior to the picture being taken.
  • The lens of the camera should be level with the drop in order to take the picture from a side-view.
  • Adjust fluorimeter height if necessary to get the drop in view of the camera.


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution Volume of the 2X DNA Solution (µL) Volume of the SYBR Green I Solution (µL) Final DNA Concentration in SYBR Green I Solution (µL)
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. Using gloves, take the slide and make sure to place it smooth side down in the fluorimeter.
  2. Turn on the fluorimeter by flipping the side switch.
  3. Pippette 80 µL of SYBR Green I Dye in between the first two clear circles on the slide (the light should be absorbed by the drop if it is in the right location).
  4. Replace pippette tip and then pippette 80 µL of the PCR Sample mix or calibration sample directly on the SYBR Green drop.
  5. Place the camera in the cradle so that it can take a side-view picture of the drop.
  6. Set the camera timer for 3-5 seconds and ensure the camera is also focused.
  7. Cover the fluorimeter box prior to the picture being taken so that no external light is present inside of it.
  8. Take at least three pictures of each sample tested to make sure that a good picture is obtained for data analysis.
  9. Open up the fluorimeter box and either replace the slide or move it so that the next two glass circles may be used (for the actual PCR Sample mix, just replace the slide)
  10. Repeat all of the steps for each sample


Data Analysis

Representative Images of Negative and Positive Samples

Positive Representative Sample

Poscontrolrepimage.jpg

Negative Representative Sample

Negcontrolrepimage.jpg

Image J Values for All Calibrator Samples


PCR Product Tube Label Area Mean Raw Int Den of Drop Raw Int Den of Back Raw Int Den Drop - Back
G# P 45816 179 8223349 27518 8195831
G# P 45923 179 8210014 21926 8188088
G# P 48080 174 8374850 41309 8333541
G# N 40712 90 3680960 17213 3663747
G# N 46105 85 3903994 27436 3876558
G# N 39024 102 3978656 21087 3957569
G# 1-1 52948 176 9317491 21087 9296404
G# 1-1 53836 179 9637205 68798 9568407
G# 1-1 49180 184 9054306 38623 9015683
G# 1-2 50012 180 8979358 28776 8950582
G# 1-2 52264 182 9500679 28776 9471903
G# 1-2 49849 183 9127715 42006 9085709
G# 1-3 55058 179 9869505 71200 9798305
G# 1-3 50632 181 9164684 69521 9095163
G# 1-3 50464 179 9023902 44497 8979405
G# 2-1 52123 77 4005829 31236 3974593
G# 2-1 57044 66 3789270 19709 3769561
G# 2-1 53736 71 3805804 20895 3784909
G# 2-2 48772 76 3710580 19685 3690895
G# 2-2 48694 73 3546428 18662 3527766
G# 2-2 51452 67 3444797 23008 3421789
G# 2-3 50240 75 3761646 22155 3739491
G# 2-3 53062 72 3844122 22155 3821967
G# 2-3 53030 68 3619168 23422 3595746


Mean Data ' ' ' '
PCR Product Tube Label Raw Int Den Drop - Back
1 2 3 Mean
G# P 8195831 8188088 8333541 8239153.333
G# N 3663747 3876558 3957569 3832624.667
G# 1-1 9296404 9568407 9015683 9293498
G# 1-2 8950582 9471903 9085709 9169398
G# 1-3 9798305 9095163 8979405 9290957.667
G# 2-1 3974593 3769561 3784909 3843021
G# 2-2 3690895 3527766 3421789 3546816.667
G# 2-3 3739491 3821967 3595746 3719068

Final Table"""

PCR Product Tube Label Y =Mean RAWINTDEN Drop - Background X =PCR Product Concentration (µg/mL) Initial PCR Product Concentration (µg/mL)
G# P 8239153.333 1.559788333 0.129982361
G# N 3832624.667 0.458156167 0.038179681
G# 1-1 9293498 1.8233745 0.151947875
G# 1-2 9169398 1.7923495 0.149362458
G# 1-3 9290957.667 1.822739417 0.151894951
G# 2-1 3843021 0.46075525 0.038396271
G# 2-2 3546816.667 0.386704167 0.032225347
G# 2-3 3719068 0.429767 0.035813917

Calibration curve

Bettercalcurveimage.jpg

PCR Results Summary

  • Our positive control PCR result was 0.13 μg/mL
  • Our negative control PCR result was 0.04 μg/mL

Observed results

  • Patient 10088 : The images of the DNA for patient 10088 did show some of the fluorescent green dye. After splitting the color using the Image J software, the green color was definitely present. The µg/mL of Patient 10088 DNA samples were all around 0.15, which was the same value as the positive control's µg/mL value of PCR Product Concentration.
  • Patient 28866 : The images of the DNA for patient 28866 showed almost none of the fluorescent green dye since the drop was almost completely blue. The color split showed that the green would not show up as clearly as it did for Patient 10088. The µg/mL of Patient 28866 DNA samples were all about 0.04, which was near the negative control's µg/mL value of PCR Product Concentration.

Conclusions

  • Patient 10088 : The results show that Patient 10088 is positive, because the value for the PCR Product Concentration was very identical to the positive control value.
  • Patient 28866 : For Patient 28866, the value of PCR Product Concentration was very close to the negative control value, which means that this patient is negative.




SNP Information & Primer Design

Background: About the Disease SNP This disease SNP is linked to a certain protein known as lipoprotein lipase. Errors in this gene cause a number of problems, primarily relating to corruptions of proper heart function. The most noticeable phenotypic reaction to the mutation we were testing for is coronary heart disease, one of the number one killers of Americans. Although primarily linked to lifestyle and health choices, this is just one of the examples of genetic predispositions that may occur.

Primer Design and Testing Selection of the primer is essential in order to accurately test for the DNA segment in question. We used an online resource to match a DNA segment we designed off of a description of the mutated strain. By coding a primer that includes the mutated allele, we ensure that our test is selective. If the mutation is present, the primer is capable of binding and the PCR process can proceed, rendering a positive test.