BME100 s2016:Group11 W1030AM L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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OUR TEAM

Name: Kyle Durrant
Name: Brandon Taylor
Name: Annette Flores
Name: student
Name: student
Name: student


LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab reading was helpful to understand just how the lab needed to be setup. A few of us have experience with using the micropipettors, so we knew what the first and second stops meant already. There was some liquid left in the tubes of the DNA samples and the PCR reaction mix, and one of the reactions had slightly less liquid in it than the rest. The labeling scheme worked fine and didn't need to be changed.

Fluorimeter Procedure

Web camera set-up

For this lab a phone camera was used instead of a webcam. The phone was placed in the cradle standing nearly vertically. The flourimeter was then placed on some small plates to lift it up to the camera. The height of the flourimeter was adjusted using more or less plates so that the camera could take a picture directly on the side of the drop. The camera was then set to use a timer.



Placing Samples onto the Fluorimeter

  1. With gloves, find the smooth side of the slide and place it on the flourimeter smooth side down.
  2. Set the camera in the cradle and set the timer on it.
  3. Change the height of the flourimeter so the camera can take a picture of the side of the drop of the solution.
  4. Use 80 microliters of SYBR Green I solution and place a drop on the first 2 clear circles in the middle of the slide.
  5. Now place 80 microliters of the sample/calibration solution on top of the SYBR Green I drop.
  6. Move the slide so that the light from the flourimeter illuminates the middle of the drop and focuses on the other side.
  7. Change the distance of the camera so that it takes a close picture of the drop and remains in focus, then record that distance.
  8. Cover the setup with the lightbox and make sure the drop is in focus.
  9. Now press the timer on the camera and close the box with the other flap.



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA
0.5 DNA
0.5DNA.jpg
5DNA
5DNA.jpg
No DNA
0DNA.jpg

Calibrator Mean Values


Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND ' ' MEAN Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 17960676 17361666 17432393 17584911.67 327337.2921
2 1 C-2 18302216 18948578 13833067 17027953.67 2785663.578
1 0.5 C-3 16029494 17146613 16272576 16482894.33 587506.6519
0.5 0.25 C-4 10520769 11682263 12226176 11476402.67 871141.2724
0.25 0.125 C-5 6119828 7453912 6946432 6840057.333 673373.3786
0 0 C-6 2816767 2841332 2611419 2756506 126247.9227


Calibration curves
Potato.png Potato2.png

Images of Our PCR Negative and Positive Controls
Nosybr.png
Negative Control
Sybr.png
Positive Control


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)
G11 + 3744312.6667 -0.322241048 12 -3.866892571
G11 - 954898.6667 -0.720728762 12 -8.648745143
G11 1-1 1533861.0000 -0.638019857 12 -7.656238286
G11 1-2 1191914.3333 -0.686869381 12 -8.242432571
G11 1-3 1426572.0000 -0.653346857 12 -7.840162286
G11 2-1 1580550.3333 -0.631349952 12 -7.576199429
G11 2-2 1403742.3333 -0.656608238 12 -7.879298857
G11 2-3 1577947.3333 -0.63172181 12 -7.580661714


PCR Results: Summary

  • Our positive control PCR result was -3.866892571 μg/mL
  • Our negative control PCR result was -8.648745143 μg/mL


Observed results

  • Patient 87806 : The image showed a drops that were faintly illuminated. It also had an average of -7.013 μg/mL.
  • Patient 99993 : The images for his patient were not illuminated and had an average of -7.679 μg/mL.


Conclusions

  • Patient 87806 : This patient returned a negative results because its PCR results most resembled the negative control.
  • Patient 99993 : This patient also returned a negative result for the same reason, its PCR results also resembled the negative control.