While we were micropipetting the samples to set up the reaction, we felt that we were well prepared from watching the pre-lab videos and reading the documents. We moved the samples with ease while using the micropipetting technique. We understood the difference between the first and second stop on the pipettor with help from the pre-lab materials. The final reactions had exactly the same amount of liquid because we pipetted all of the liquid from the samples that were given to us. There was no liquid left in the tubes that the DNA samples and PCR reaction mix, which means that we pipetted the same amount of each. We did not have to change our labelling scheme because our system allowed for clear differences between the samples.
Fluorimeter Procedure
Imaging set-up
While we were setting up our device to capture the images from the fluorimeter, we stared with turning on the fluorimeter and placed it on the table. Then, we placed the slide in the fluorimeter with the sooth side down. After that, we adjusted the height of the fluorimeter to the height of the camera in order to capture a proper image using a camera timer set for 3 seconds on a smartphone.
Placing Samples onto the Fluorimeter
The first step is to identify two clear circles on the middle of the slide where we will then place 80 uL drop of SYBR Green 1 solution.
Step two is to the place 80 uL drop of sample/calculation that will mix with the SYBR Green Solution 1.
Then, make sure that the blue light lights up the center of the drop by moving the slide accordingly.
Making certain that the phone is more than 4 centimeters away from the drop, record the distance and ensure that you are able to have a clear, consise view of the drop thorugh the lens.
After that, place the light box with the open side facing the lab group, and double check that the drop is focused with the camera an additional time before the picture is taken.
After the picture is taken, remove the drop from the slide and discard the waste into a liquid waste container. Lastly, move the slide to the next row of two circles for the next drop to be analyzed.
Repeat steps 1-6 for all samples.
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)
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
5
2.5
C-1
135097
119824
111796
122239
11836.73599
2
2
1
C-2
107735
111997
107583
109105
2505.698306
1
1
0.5
C-3
79634
83274
77511
80140
2914.58682
0.5
0.5
0.25
C-4
77443
76321
79551
77772
1639.890647
0.25
0.25
0.125
C-5
69680
79559
68328
72522
6131.311795
0
0
0
C-6
C-6
0
0
0
0
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control
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)"
G10+
3477766
322.2470889
12
3866.965067
G10-
1135421.667
100.4971757
12
1205.966108
G10 1-1
2557789
235.1527975
12
2821.83357
G10 1-2
2555368.667
234.9236644
12
2819.083973
G10 1-3
3234299
299.1980498
12
3590.376598
G10 2-1
1110961.667
98.18154568
12
1178.178548
G10 2-2
687185.3333
58.06260847
12
696.7513017
G10 2-3
538257
43.96355202
12
527.5626243
PCR Results: Summary
Our positive control PCR result was 3866.965067 μg/mL
Our negative control PCR result was 1205.966108 μg/mL
Observed results
Patient 72874: Qualitatively, the images taken for this patient in the fluorimeter showed a bright green color within the droplet of SYBR Green I and sample. The average in concentration for patient 1 was closer to the control positive control.
Patient 64687: The images for this patient showed a very faint/dark green color within the SYBr Green 1 solution droplet. The average in concentration for this patient was also closer to its negative control value.
Conclusions
Patient 72874: This patient tested positive because its concentration tested closer to the positive control value, and illuminated green within the SYBR Green 1 solution.
Patient 64687: This patient tested negative because its concentration was closer to the negative control value and did not illuminate green at all in the SYBR Green 1 solution.
BME 100 Fall 2017
