Out of the entire group Lauren and Talia were the only ones to have used micropipettors before. To help everyone feel more comfortable, Lauren and Talia worked with pipetting the solutions into the proper test tubes. The pre-lab reading definitely helped refresh the steps and made it easier to carry out the proper procedure. We did know the difference between the first and second stops after testing out the micropipettor a couple times. From observations, each test tube seemed to have as close of a reading as possible to the 100 μl. In the DNA sample test tubes and PCR reaction mix test tubes, there was a little bit of solution left in them. Our labeling scheme was our group number, then the labels of; positive, negative, patient 1-1, patient 1-2, patient 1-3, patient 2-1, patient 2-2, patient 2-3. While pipetting, we did not need to change our scheme because it worked out perfectly.
Fluorimeter Procedure
Imaging set-up
The materials that we gathered were a fluorimeter, about 10 glass slides, a phone stand for the smartphone, and a light box.We used our teammate's iPhone, of which required us to raise the light box up with about 4 carton boxes so that it was properly aligned with the camera on the phone. We also made sure that the phone was close enough to the droplet to get a clear image, so we remained about 4 cm away from the drop.This allowed us to see the appropriate solution of our PCR reaction mixed with the SYBR Green 1 solution on the glass slide. Then, the phone was set to have a 3 second timer for taking the pictures and the flash was turned off. So when the pictures needed to be taken, we just pressed the capture button on the phone and quickly lowered the lid of the light box. Then, our pictures were labeled with the right trial and saved to the phone to be later uploaded on ImageJ.
Placing Samples onto the Fluorimeter
1. Place a 80 microliter drop of SYBR Green 1 on top of the 80 microliter drop of one of the calf thymus solutions (these drops will be placed in the middle of the first 2 rows of the slide)
2. Align the drop so that the blue LED light is focused from one side of the drop to the other
3. Place phone on stand and press capture button while immediately closing lid of light box
4. Take 3 pictures of the droplet and make sure the flash is off and the image is focused
5. Remove the box. Be careful to not adjust the position of the phone too much
6. Take micropipettor and remove the 160 microliter drop from the slide and prepare for new sample (you can always take a new slide if needed)
7. Repeat steps 1-6 for all the other concentrations of calf thymus solution
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
0 μg/mL sample
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
RAWINTDEN DROP - BACKGROUND
RAWINTDEN DROP - BACKGROUND
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
19607443
27267726
26689551
24521573.33
4265569.046
2
1
C-2
15769374
15861018
165006363
65545585
86135572.62
1
0.5
C-3
16840698
14789405
17830034
16486712.33
1550914.43
0.5
0.25
C-4
16626467
16080101
15705150
16137239.33
463308.5856
0.25
0.125
C-5
12399828
12182004
11214249
11932027
631083.0109
0
0
C-6
5559981
6217025
6217025
5998010.333
379344.5303
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive PCR Mix
Negative PCR Mix
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)
G10:Positive
16662422
0.4887474
12
5.8649688
G10:Negative
4070948.333
0.0690316111
12
0.8283793332
G10:1-1
26116407.33
0.8038802443
12
9.646562932
G10:1-2
19016194.67
0.567206489
12
6.806477868
G10:1-3
15533066.33
0.451102211
12
5.413226532
G10:2-1
7755593.333
0.1918531111
12
2.302237333
G10:2-2
1011421
-0.03295263333
12
-0.3954316
G10:2-3
7767009.667
0.1922336556
12
2.306803867
In the table above, the first 1 represents Patient 1 (28319) and the first 2 represents Patient 2 (81362). The second number that immediately follows the dash represents the replicate number. For example, G10:1-1 represents Patient 1 (28319) replicate number 1.
PCR Results: Summary
Our positive control PCR result was 5.8649688 μg/mL
Our negative control PCR result was 0.8283793332 μg/mL
Observed results
Patient 28319 : Qualitatively, in the pictures of our first patient we clearly saw a bright green color in all three replicates. The three PCR observed product concentrations were 9.646562932 μg/mL, 6.806477868 μg/mL, 5.413226532 μg/mL.
Patient 81362 : Qualitatively, in the pictures of our second patient we did not see any green glowing color in any of the three replicates. The three PCR observed product concentrations were 2.302237333 μg/mL, -0.3954316 μg/mL, 2.306803867 μg/mL.
Conclusions
Patient 28319 : Based off both of our qualitative and quantitative analysis of our results, we can conclude that patient 28319 yields a positive result. This can also be seen by comparing the PCR Product concentrations to our controls values. The values of patient 28319 most closely compared to our positive control value of 5.865 μg/mL.
Patient 81362 : Based off both of our qualitative and quantitative analysis of our results, we can conclude that patient 81362 yields a negative result. This can also be seen by comparing the PCR Product concentrations to our controls values. The values of patient 81362 most closely compared to our negative control value of 0.828 μg/mL.
BME 100 Fall 2018
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