BME100 f2017:Group6 W0800 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: Tajinder Uppal
Name: Tim Oetter
Name: Joshua Gunn
Name: Trevor Heaton
Name: Jesus Dorame


LAB 5 WRITE-UP

PCR Reaction Report

While pipetting, our group ran into no problems, it took a couple trials to get the hang of things but after that everything went smoothly. The pre-lab reading gave a great overview of what to expect. We were able to understand the difference between the first and second stop soon after getting the pipettor. As far as the final reactions, we had the same amount of liquid for each product. Also, we extracted all the liquid from the tubes so there was no liquid left in the DNA samples or the PCR reaction mix. Lastly, we were able to keep our labeling scheme the same as last the lab.

Fluorimeter Procedure

Imaging set-up

Samsung S8
  1. Go to camera menu
  2. Deactivate the flash
  3. Set ISO to 800
  4. Set White Balance to Auto
  5. Set exposure to highest setting
  6. Set Saturation to Highest setting
  7. Set contrast to the lowest setting
  8. Set Timer on phone
  9. Have camera on three shot mode
Slide and Phone Position setup
  1. Set phone base about 6 cm away from slide
  2. Make sure the image is at a 90 degree angle view
  3. Slide must be level with camera lens
  4. After starting camera timer, cover with given box to reduce light exposure


Placing Samples onto the Fluorimeter

  1. First, as a control, use the micropipette to place 160 microliters of water onto the slide
  2. Use smartphone to capture three images of water droplet
  3. Use micropipette to extract the water from the slide
  4. Use micropipette to distribute 80 microliters of SYBR GREEN 1 solution onto the slide
  5. Add an additional 80 microliters of the 0.25 concentration Calf Thymus DNA Solution onto the slide
  6. Use smartphone to capture three images of droplet
  7. Extract liquid from the slide using micropipette
  8. Repeat steps 3 through 7, except instead of adding the 0.25 concentration Calf Thymus DNA solution, add the solution of interest (0.5, 1, 2, 5, Positive Control, Negative Control, 1-1, 1-2, 1-3, 2-1, 2-2, 2-3)


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Zero Calf Thymus DNA 0 1 OWW.jpg

Low Calf Thymus DNA 0.5 1 OWW.jpg

High Calf Thymus DNA 5.0 1 OWW.jpg


Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution Final DNA concentration in SYBR Green I solution (μg/mL) Sample Number RAWINTDEN DROP - BACKGROUND RAWINTDEN DROP - BACKGROUND RAWINTDEN DROP - BACKGROUND Mean Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 3850171 3733338 3558373 3713960.667 146860.9172
2 1 C-2 4549335 4602907 5067319 4739853.667 284855.4865
1 0.5 C-3 3916289 3598140 2612292 3375573.667 679892.5456
0.5 0.25 C-4 3010475 3300069 3147165 3152569.667 144872.6304
0.25 0.125 C-5 2679559 2140249 2722682 2514163.333 324536.3536
0 0 C-6 1958892 1956585 1961565 1959014 2492.240558


Calibration curves

DP 1.PNG

DP 2.PNG


Images of Our PCR Negative and Positive Controls

Positive Control Positive Control 1 OWW.jpg

Negative Control Negative Control 1 OWW.jpg


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP-BACKGROUND) PCR Product Concentration (µg/mL) Total Dilution Initial PCR Product Concentration (µg/mL)
G6+ 3604229 1.604 12 19.248
G6- 2289819 0.29 12 3.48
G6 1-1 1663413 -0.337 12 -4.044
G6 1-2 2506477 0.506 12 6.072
G6 1-3 2619408 0.619 12 7.428
G6 2-1 3001456 1.001 12 12.012
G6 2-2 2163073 0.163 12 1.956
G6 2-3 2329024 0.329 12 3.948


PCR Results: Summary

  • Our positive control PCR result was 19.248 μg/mL
  • Our negative control PCR result was 3.48 μg/mL


Observed results

  • Patient 40032 : All of the pictures taken of this patient's DNA showed very little concentration because the green dye was hardly fluorescent. This is proven by the average concentration of about 6 μg/mL.
  • Patient 98170 : All of the pictures taken of this patient's DNA showed very little concentration because the green dye was hardly fluorescent. This is proven by the average concentration of about 4 μg/mL.


Conclusions

  • Patient 40032 : This patient is very similar to the negative control. This patient does not have the SNP associated with the disease.
  • Patient 98170 : This patient is very similar to the negative control. This patient does not have the SNP associated with the disease.