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OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The pipetting of samples to set up the polymerase chain reaction was relatively successful. The pre-lab reading helped remind us how to properly use a micropipettor and the procedure necessary for it. The difference between the first and second stop was easy to feel on the pipettor. The final reactions should have had the same amount of liquid, but due to some amount of user error, there were most likely less liquid in some of the reaction tubes than others. There was liquid left in the tubes that contained the DNA samples and PCR reaction mix, but not much. We did not have to change our labeling scheme, as it made logical sense and was easily understandable.
Fluorimeter Procedure
Imaging set-up
The team had to set up one of the smartphones to capture images from the fluorimeter. This was done by inactivating the flash, setting the ISO to 800, and putting the white balance on the auto setting. We were unable to change the exposure, saturation, and contrast on the phone. Afterwards, the smartphone was placed in the cradle and the fluorimeter was raised up on pipette tip boxes so that the camera would take the picture of the drop edge-on. The camera was also focused on the drop to ensure a proper photo. The smartphone was also set to take three pictures consecutively, after a 5-second timer, so that the photos could be taken inside the light box.
Placing Samples onto the Fluorimeter
- Set the micropipettor to 120 microliters so that all 100 microliters of the PCR sample are drawn up.
- Attach a disposable pipette tip from the tray to the micropipettor and transfer all of the volume from the PCR sample tube into the 500 microliter buffer tube for each PCR sample, discarding the micropipette tip in between each, effectively diluting your samples.
- Close the cap of the buffer tube and invert the tube to mix the two solutions.
- Take the micropipettor and set it to draw up 80 microliters.
- Use the micropipettor to draw up 80 microliters of the SYBER Green I solution and place the drop onto the middle circle of the rough superhydrophobic side of the glass slide in between the first two rows of the slide.
- Discard the tip and get a new pipette tip to draw up 80 microliters of the PCR sample solution and place the solution on top of the drop of SYBER Green I.
- After the pictures are taken of the solution, switch the micropipettor to 160 microliters in order to remove the drop from the glass slide.
- Repeat steps 4 through 7 for all PCR-Buffer solutions.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
- 5 μg/mL sample
- 0.5 μg/mL sample
- zero DNA
Calibrator Mean Values
| Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
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Final DNA concentration in SYBR Green I solution (µg/mL)
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Sample Number
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RAWINTDEN DROP - BACKGROUND
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'
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'
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MEAN
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Standard Deviation
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Image 1 |
Image 2 |
Image 3 |
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| 5 |
2.5 |
C-1 |
13573365 |
12659440 |
12122671 |
12785159 |
733472.7
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| 2 |
1 |
C-2 |
8404299 |
8759178 |
8949307 |
8704261 |
276623
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| 1 |
0.5 |
C-3 |
8447202 |
8114073 |
9149635 |
8570303 |
528642.2
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| 0.5 |
0.25 |
C-4 |
7020236 |
6706053 |
7600584 |
7108958 |
453817.2
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| 0.25 |
0.125 |
C-5 |
6580519 |
6744682 |
6190122 |
6505108 |
284867.3
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| 0 |
0 |
C-6 |
1087474 |
941842 |
1083411 |
1037576 |
82932.67
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Calibration curves
Images of Our PCR Negative and Positive Controls
- Negative Control PCR Sample
- Positive Control PCR Sample
PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL
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MEAN (of RAWINTDEN DROP - BACKGROUND)
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PCR Product Concentration (µg /mL) (Step 5 calculation)
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Total Dilution
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Initial PCR Product Concentration (µg /mL) (Step 6 calculation)
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| G9 + |
12945820.33 |
2.98194011 |
12 |
35.78328132 |
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| G9 - |
2736661 |
-0.421113 |
12 |
-5.053356 |
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| G9 1-1 |
8019664.667 |
1.339888222 |
12 |
16.07865867 |
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| G9 1-2 |
9475502.667 |
1.825167556 |
12 |
21.90201067 |
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| G9 1-3 |
4710894.333 |
0.2369647777 |
12 |
2.843577332 |
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| G9 2-1 |
3427914.333 |
-0.1906952223 |
12 |
-2.288342668 |
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| G9 2-2 |
2801499.333 |
-0.3995002223 |
12 |
-4.794002668 |
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| G9 2-3 |
2145861 |
-0.6180463333 |
12 |
-7.416556 |
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PCR Results: Summary
- Our positive control PCR result was 35.78328132 μg/mL
- Our negative control PCR result was -5.053356 μg/mL
Observed results
- Patient 20925: Patient 20925 comprised of our G9 1-1, 1-2, and 1-3 data set. The images looked bright and white in comparison to the negative control PCR result and were more similar to the positive control PCR result. 1-1 and 1-2 were brighter than 1-3, however, this is most likely due to some kind of error in lab procedure. The micrograms/mililiter calculated as the initial PCR concentration values were 16.07865 μg/mL, 21.90201067 μg/mL, and 2.843577332 μg/mL respectively for 1-1, 1-2, and 1-3.
- Patient 23743: Patient 23743 comprised of our G9 2-1, 2-2, and 2-3 data set. The images were dark and grey in comparison to the positive control PCR result, which was bright and white, so that the images were more similar to the negative control PCR result. The calculated initial PCR concentration values for 2-1, 2-2, and 2-3, respectively, were -2.288342668 μg/mL, -4.794002668 μg/mL, and -7.416556 μg/mL.
Conclusions
- Patient 20925: This patient's results were closer to the positive control value, as the average of the initial PCR concentration values calculated for the patient was 13.60808222 μg/mL, and the positive control PCR result concentration was 35.78328132 μg/mL. The calculated initial concentration values for this patient were all positive as well, in comparison with the negative result found in the negative control concentration, while the positive control value gave a positive result. As a result, the conclusion drawn from Patient 20925 is that it tested positive for the disease SNP that we were trying to detect because the concentration of the DNA sequence we were looking for was a substantial positive value.
- Patient 23743: This patient's results were closer to the negative control value, as the average of the initial PCR concentration values calculated for this patient was -4.832967112 μg/mL, and the negative control PCR result concentration was -5.053356 μg/mL. The calculated initial concentration values for this patient were all negative as well, which matched the negative result of the negative control concentration. As a result, the conclusion drawn from Patient 23743 is that it tested negative for the disease SNP that we were trying to detect because the concentration of the DNA sequence we were looking for was a small negative value.
- The negative control PCR result and Patient 23743 had negative calculated initial PCR concentration values most likely due to some kind of error in the lab procedure when creating the calibration curve. The initial PCR concentration values depended on using the calibration curve, and so the negative numbers indicate that the mean RAWINTDEN of the drop minus the background of the negative control and Patient 23743 were lower than the means of the calf thymus samples.
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BME 100 Fall 2017
