BME100 f2017:Group11 W0800 L5

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Our Team

Name: Braeden Malotky
Name: Mauro Robles
Name: Alexa Ng
Name: Keiko Ochoa
Name: Connor Leicken

PCR Reactions

Two patients have each submitted three replicate DNA samples to be tested for a disease marker. The six samples, in addition to one positive and one negative control, were analyzed. To test for the disease marker, each sample of DNA was mixed with a PCR reaction mix and primers, which target the specific piece of DNA associated with the disease. The eight samples were then placed in the thermocycler to amplify the DNA. The positive control and any of the samples of the patients with the disease should have high frequency of DNA because the primer was able to find and attach to the disease DNA section, thus amplifying it. There should be a low amount of DNA amplified in the negative control and the patients' samples without the disease, because the primer was unable to attach to the disease-associated DNA piece and amplify it.

Fluorimeter Procedure

Camera Set-Up

Model: iPhone 7

1. Change settings on camera

  • Turn off flash
  • Set ISO to 800
  • Set Exposure to highest setting (2)
  • Set Saturation to highest setting (if possible)
  • Set Contrast to lowest setting (if possible)

2. Set camera timer to three seconds

3. Set the camera in the cradle so that the drop on the fluorimeter slide is in focus and that the camera is as close to the drop as possible

4. Measure the distance between the fluorimeter and the camera. Maintain constant distance for every photo. (Ours was 4cm)

5. Cover the entire system (fluorimeter and camera) with the box so the picture is taken in relative darkness

Using the Fluorimeter

1. Turn on the fluorimeter so that the blue light is on

2. Place the slide into the fluorimeter smooth side down.

3. Place a 80 microliter drop of SYBR GREEN I on the first two holes in the middle rows of the slide using the micropipettor. Using a new micropipettor tip, then add 80 microliters of either the calf thymus (or water blank) solutions.

4. Take three pictures of the drop using the smartphone camera procedure described above. Make sure to document the images correctly afterwards.

5. Remove the drops from the slide using the micropipettor. Move the slide forward so the next two holes are at the front and repeat steps 2-5 for the next DNA sample.

Data Analysis

Table 1: PCR Raw Data

Sample Number Image Number Final DNA Concentration Area Mean Pixel Value RawIntDen of the Drop RawIntDen of the Background Minimun Maximum Color
1 1 0.125 46504 135.441 6298558 175878457 25 255 None
1 2 0.125 42528 127.022 5401981 165393967 37 255 None
1 3 0.125 47044 143.201 6736742 197932480 48 255 None
2 1 0.25 69476 131.232 8828402 1890958259 8 255 Green
2 2 0.25 65444 143.902 9417545 181485250 32 255 Green
2 3 0.25 62180 139.062 8645893 186035957 11 255 Green
3 1 0.5 73968 158.401 11716623 219467452 27 255 None
3 2 0.5 80252 158.168 12693271 200502424 43 255 None
3 3 0.5 80185 150.751 12087944 199536625 33 255 None
4 1 1 105792 128.667 13611992 134825536 15 255 None
4 2 1 116248 125.272 14562663 126905865 15 255 None
4 3 1 114692 128.927 14786851 128998070 14 255 None
5 1 2.5 108572 119.377 12960969 147753261 27 255 None
5 2 2.5 103184 116.355 12005943 141339941 19 255 None
5 3 2.5 107328 117.294 12588935 140756949 20 255 None
6 1 0 80596 154.013 12729926 109203822 39 255 Green
6 2 0 123208 151.694 18689945 128893087 30 255 Green
6 3 0 73032 154.514 11284471 99827747 48 255 Green
7 1 0 137412 86.805 11928032 166099184 18 255 None
7 2 0 135032 87.46 11809964 158043551 19 255 None
7 3 0 135284 90.478 12240209 167824147 10 255 None
8 1 0 82360 142.918 11823742 177234556 17 255 None
8 2 0 86848 148.175 12868732 176300973 20 255 None
8 3 0 71268 148.344 10572213 17866576 55 255 None

Pictures of Thymus DNA at 0,0.25, and 1.0

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