The lab group first micropipetted 50 microliters (µL) of each individual DNA/primer mix into properly labeled tubes. Taking care not to cross contaminate each sample mix, another 50 µL of PCR reaction mix was added and mixed with the DNA/primer solutions. Pipette tips were carefully disposed before each individual solution. During the first three mixes, the micropipetter was new to the technique and could have been a source of error for measurement.
These tubes were then placed in the PCR machine and run through 25 cycles. The lid was set to 100°C. The initial step was set to 95°C for two minutes. The cycles were set for 30 seconds each to denature at 95°C, anneal at 57°C and extend at 72°C. The computer connected to the PCR machine logged out the user after 10 minutes, and although this did not appear to affect the machine it could have caused error or a glitch in the cycles.
A few members in our group have had experience with pipetting so they were able to help everyone during the lab. The pre-lab helped because the simulation for the pre-lab was very clear and detailed enough to help understand what we would be doing in the lab. We understood the difference between the first and second stop on the pipettor very easily because of the pre-lab simulation. We used it as guidance during the lab. The final reaction did not have the same amount of liquid due to air bubbles in the solution and transfer process, most likely caused by human error. Additionally, there was not any excess liquid left in the tubes that the DNA samples and PCR reaction were contained in. We did not have to adjust or change the labeling system that we established in the previous lab.
Fluorimeter Procedure
Imaging set-up
To properly capture images from the fluorimeter, a smartphone camera needs to be set up. To make sure the Camera is at the same level of the beam, try adding support underneath the device to raise it. Click on the timer on the camera app and set the timer to at least 3 seconds. Capture each image after lowering down the end flap to eliminate any exterior light coming through.
Placing Samples onto the Fluorimeter
[Step one: Find the smooth side of the slide. ]
[Step two:Turn on the Fluorimeter and place the slide in the device smooth side down. ]
[Step three: Set up the camera with a 3-second timer on the cradle and adjust the height of the device to make sure the droplet is in perfect view.]
[Step Four: Place 80 uL drop of SYBR green solution in the middle of the slide towards the top edge (first two bubbles). ]
[Step Five: Place 80 uL drop of Calibration sample on top of the SYBR green solution. Make sure the slide is adjusted so the droplet is in the center of the beam. ]
[Step Six: Record the distance of the Camera from the Device. Cover the Device with the black box, with one flap open for easy access. ]
[Step Seven: Take a picture and close the end flap before the shutter takes the snapshots. ]
[Step Eight: After taking 3 different pictures, remove the 160 uL solution and dispose in the liquid disposal. Move the slide up to the next row of circles. ]
[Step Nine: Repeat the above steps until you have used all 5 measurement positions on the slide. ]
Data Collection and Analysis
Image of High Calf Thymus DNA
Image of Low Calf Thymus DNA
Image of Zero Calf Thymus
Calibrator Mean Values
Sample Number
Image Number
Final DNA concentration in SYBR Green I solution (µg/mL)
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAW INTDEN OF THE BACKGROUND
RAWINTDEN DROP-BACKGROUND
C-1
1
2.5
5728
165.1
945693
17111
928582
C-1
2
2.5
5054
166.689
842447
19233
823214
C-1
3
2.5
5586
165.741
925830
21179
904651
C-2
1
1
5646
137.762
777807
30685
747122
C-2
2
1
14195
145.592
2066676
131606
1935070
C-2
3
1
14130
158.958
2246077
74551
2171526
C-3
1
0.5
5206
155.018
807026
23160
783866
C-3
2
0.5
5206
145.66
758305
36886
721419
C-3
3
0.5
5689
140.589
799810
46994
752816
C-4
1
0.25
8960
129.482
1160158
99554
1060604
C-4
2
0.25
8079
135.419
1094049
81740
1012309
C-4
3
0.25
23044
120.623
2779640
215749
2563891
C-5
1
0.125
9260
117.697
1089877
102904
986973
C-5
2
0.125
22062
117.001
2581284
228167
2353117
C-5
3
0.125
20962
120.203
2519702
217999
2301703
C-6
1
0
7240
82.913
600292
26087
574205
C-6
2
0
7317
76.106
556867
24329
532538
C-6
3
0
6908
69.069
477128
41917
435211
Calibration curves
Images of Our PCR Negative and Positive Controls
Image of Positive Control
Image of Negative Control
PCR Results: PCR concentrations solved
PCR Product Concentration (µg /mL)
Total Dilution
"Initial PCR Product Concentration
(µg /mL)
4.42696942
12
53.12363304
0.667358087
12
8.008297048
-0.030511217
12
-0.366134599
0.006802175
12
0.081626101
0.347821398
12
4.173856773
0.311751309
12
3.741015711
0.259756807
12
3.117081686
2.096657604
12
25.15989125
PCR Results: Summary
Our positive control PCR result was 53.120 μg/mL
Our negative control PCR result was 8.010 μg/mL
Observed results
Patient 27468: After observing the droplet under the ImageJ software, we noticed a strong appearance of green towards the center of the drop. 4.17 μg/mL was observed for Patient 27468.
Patient 96944: While observing the droplet during the experiment, The drop for Patient 96944 showed no difference in appearance (naked eye observation) as compared to the Patient 27468 droplet. 25.16 μg/mL was observed for Patient 96944.
Conclusions
Patient 27468: Negative___: We concluded the patient was negative based on the values that matched our negative control.
Patient 96944:Negative___: We concluded the patient was negative based on the values that matched our negative control.
BME 100 Fall 2016
