BME100 f2015:Group9 1030amL5

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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: Glenna Bea Embrador
Name: Ian Quinn
Name:Eric M Rhoades
Name: Chase Gregor
Name: Broderick T. Laese
Name: Nimisha Tanna


LAB 5 WRITE-UP

PCR Reaction Report

Summary of Conducting PCR Reaction
Pipetting the samples for the reaction went smoothly after doing the pre-lab reading because it allowed us to familiarize ourselves with the materials and the methods in pipetting with the most accuracy and precision. Before actually conducting the lab, the tubes are labeled using the data from Lab Report 4, and the samples are thoroughly thawed in order to transfer the mixture into the labeled tube. After, the first step in conducting the lab is transferring the PCR mixture to the tube labeled G9 +. To do this, the micropipette is set to 50μL and a tip is attached to the micropipette. The plunger is then pressed to the first stop prior to placing the tip into the tube with the mixture. Pressing the plunger to the first stop allows the precise amount of volume to be ready to draw in the liquid specified on the micropipette. After, the tip is placed in the empty labeled tube and the plunger is slowly pressed to the second stop, which dispenses the liquid in the micropipette. These steps were repeated (changing the tip each time) with the specified DNA samples and controls. On one transfer, there was an air bubble in the micropipette which prevented all the liquid being dispensed into the labeled tube. In order to fix this, the liquid was drawn again and placed back into the tube in order to dispense all the liquid. After each tube is filled, it is then placed into the thermal cycler where it begins to denature and replicate as part of the PCR reaction.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 6
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Camera set-up

  1. Use the provided cradle to position the phone at least 4 cm away from the fluorimeter
  2. Place the phone in the phone stand and adjust accordingly in order to get the camera to capture a side view of the fluorimeter
  3. Place an empty container underneath the fluorimeter in order to level the device with the camera, in this case a short plastic container is used
  4. Make sure everything is stable in order to get the ideal image of the fluorimeter.

  • Distance between the smart phone cradle and drop = 4 cm


Placing Samples onto the Fluorimeter

  1. First, place a clean side onto the fluorimeter with the smooth side down
  2. Next, (using proper pipetting techniques) pipette 80 µL of SYBR Green I solution and release the drop on the slide between the first two circles in the center of the slide.
  3. Then, pipette 80 µL of the sample solution, and add it to the SYBR Green I drop
  4. Turn on the light of the fluorimeter and adjust the light so that it is focused through the other side of the drop
  5. Make sure the smartphone that is being used to take pictures (and the cradle it is in) is more than 4 cm away from the fluorimeter. Record this distance.
  6. Once the smartphone is positioned correctly, cover the phone and the fluorimeter entirely with the lightbox and take a picture
  7. The drop is then removed from the slide via the micropipette and is disposed of in the proper container
  8. The steps 1-6 is repeated every sample, moving by two circles for each one


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Water Zero DNA.png
0 Calf Thymus DNA; Raw Image
WATERWCIRCLEgroup5.png
0 Calf Thymus DNA in ImageJ; With circle around droplet
Point5fullcolorgroup5.png
0.5 μg/mL sample Calf Thymus DNA;
Raw Image
Point5group5.png
0.5 μg/mL sample Calf Thymus DNA in ImageJ;
With circle around droplet
5 GRouuUP five.png
5 μg/mL sample Calf Thymus DNA;
Raw Image
5 DNA GROUP 5.png
5 μg/mL sample Calf Thymus DNA in ImageJ;
With circle around droplet


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 16932397 17326457 16889295 17049383 240918.9629
2 1 C-2 15164678 15449211 16063478 15559122.33 459369.9336
1 0.5 C-3 14328862 14500951 14634062 14487958.33 153014.2707
0.5 0.25 C-4 15501228 15468328 15431230 15466928.67 35019.97432
0.25 0.125 C-5 11867903 12050523 11632013 11850146.33 209819.278
0 0 C-6 12437044 12361200 12330057 12376100.33 55027.8974


Calibration curves

Dotplot1-2.PNG

Dotplot2.PNG


Images of Our PCR Negative and Positive Controls

Glennaplusok niceu.png
Positive control PCR sample;
Raw Image
G+ group 5 glenna e.png
Positive control PCR sample;
With circle from Image J
G CoNTROl negative Group 5 1030.png
Negative control PCR sample;
Raw Image
G MINUS GROUP 5 image j.png
Negative control PCR sample;
With circle from Image J


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)
G+ 196.926 1.7074269 12 28.5708
G- 134.328 0.9064451 12 3.30532
G 1-1 229.602 1.5448568 12 3.6623
G 1-2 205.664 1.4697539 12 1.69084
G 1-3 237.445 1.72442686 12 4.06962
G 2-1 210.613 1.5858216 12 1.70422
G 2-2 201.241 1.4999670 12 1.5326
G 2-3 229.24 1.6483451 12 2.19837


PCR Results: Summary

  • Our positive control PCR result was 28.57 μg/mL
  • Our negative control PCR result was 3.31 μg/mL


Observed results

  • Patient 14176: When tested through the flurometer, slight pigments of green were relevant in the image taken.
  • Patient 91779: When tested through the flurometer, more concentration of green pigment is relevant.


Conclusions

  • Patient 14176 : Due to the green pigment in the sample, the patient is inflicted with the disease. The patient is more or less on the positive end of the control and will express the single nucleotide polymorphism.
  • Patient 91779 : Due to the green pigment in the sample, the patient is inflicted with the disease. The patient is more or less on the positive end of the control and will express the single nucleotide polymorphism.