Overall, we felt that we had a pretty thorough understanding of what was going on during the PCR labs. The pre-lab materials and quizzes definitely encouraged us to prepare and helped us test our knowledge as to whether or not we were prepared enough for the lab that day to ensure that everything would run smoothly. Initially, we were a little confused because of the complexity of the lab; however, after coming to lab and having the professors further demonstrate and discuss the procedures, everything began to clear up. Using the micropipettes was a great experience, and it was easy to understand the difference between the first and second stops and their respective uses. The final reactions did have the same amount of liquid, and there was liquid left over in our PCR tubes containing the DNA samples. We did not have to change our labeling scheme because we felt that our labels were unique enough not to get confused with those of another group.
Fluorimeter Procedure
Imaging set-up
To make the fluorimeter level with the camera on the phone, our team set up the fluorimeter on top of a stack of micropipette tip boxes. The device we used to take photos was an iPhone 7 with a silicone phone case on the iPhone to make it easier to stand the phone up perpendicular to the table. Placing the phone in a black phone stand, we then placed the phone approximately six centimeters away from the fluorimeter. With each new droplet, three photos were taken by touching the screen of the phone without moving the phone or the stand that the phone was in. The images were then automatically uploaded to a MacBook Pro, and processed in ImageJ.
Placing Samples onto the Fluorimeter
First, the light on the fluorimeter was turned on, and the slide was adjusted so that the blue light was shining on the section of the slide where the bubble was going to be dispensed.
Using the micropipette with a fresh tip, 160μL of diluted DNA solution was collected and dispensed in the middle of the first two rows of the slide with the rough side facing up, making sure to dispense the liquid perpendicular to the slide to ensure that the bubble of liquid formed as round as possible.
Again, using the micropipette with a fresh tip, 80μL of SYBR Green was dispensed directly on top of the bubble of DNA solution.
The black cover box was then placed over the top of the fluorimeter and the iPhone 7 so that the bubble's color change would appear more easily and three photos could be taken.
After three photos were taken, the box was then removed, and the liquid on the slide was collected into the micropipette using a fresh tip and was then disposed of properly.
Before each new sample was placed on the slide, the slide was moved back two rows to ensure that the new samples were not contaminated by the previous samples.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Zero DNA:
0.5 μg/mL Sample:
5 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
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
14712456
15474412
13812527
14666465
831896.5184
2
1
C-2
10257005
9574465
9531912
9787794
406905.2855
1
0.5
C-3
9486730
9449195
9645957
9527294
104464.8253
0.5
0.25
C-4
7235892
7545092
7438104
7406362.667
157024.8203
0.25
0.125
C-5
5711918
5752881
5884094
5782964.333
89943.86556
0
0
C-Water
3925245
3588584
3930734
3814854.333
195975.0752
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control:
Negative 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)
PC3
14859721.67
3.28657389
0.08
0.26
NC3
10477835.33
1.82594511
0.08
0.146075609
P1-1
7909878.667
0.969959556
0.08
0.077596764
P1-2
4600496.333
-0.133167889
0.08
-0.010653431
P1-3
2951348.333
-0.682883889
0.08
-0.054630711
P2-1
2592101.333
-0.802632889
0.08
-0.064210631
P2-2
5706986.667
0.235662222
0.08
0.018852978
P2-3
5484116.333
0.161372111
0.08
0.012909769
PCR Results: Summary
Our positive control PCR result was 0.26 μg/mL
Our negative control PCR result was 0.14607561 μg/mL
Observed results
Patient 74627: For our first patient, upon first glance, no color change was noticed to the naked eye. After taking photos of the bubble, there was still no color change noticed. With regard to the numbers, the Initial and Final PCR Product Concentrations of this patient were both much closer to the value of the negative control.
Patient 11603: For our second patient, upon first glance, there was not a color change observed to the naked eye either, and none still after the photos were taken. With regards to the numbers, again the Initial and Final PCR Product Concentrations of this patient were both closer to the values for the negative control, but they were farther from the negative control values than our first patient.
Conclusions
Patient 74627: In comparison to the negative and positive controls, this patient is definitely more comparable to the negative control. Starting with the photos taken for patient one, they look much more similar to the negative control in terms of how much CYBR Green was visible throughout the solution. In terms of numbers, the numbers for this patient were significantly less than that of the negative control, yet still closer to the negative control than the positive control. This indicates that this patiently most certainly does not have the disease, but could possible carry it as a recessive trait. However, the numbers are so low that it is unlikely that this patient will contract or have symptoms of this disease at all.
Patient 11603: In comparison to the negative and positive controls, this patient was again comparable to the negative control. Again, the photos were more comparable to the negative control, but in terms of numbers, the Initial and Final PCR Product Concentrations for this patient were more than significantly lower than the negative control. This could indicate that not only does this patient not have the disease, but the likeliness that they carry any genes or mutations within their DNA that would cause them to have symptoms or develop the disease in the future was not very likely.
BME 100 Fall 2017
