When pipetting the samples, our group did not run into any issues. This was in part due to the pre-lab reading, which explained in detail on how we were to go about pipetting the samples without making errors. The pre-lab reading also helped us understand the PCR process and allowed us to calibrate the PCR device, which included the steps and temperatures of each step of the PCR process. The first stop of the pipettor was to vacate air and to prepare for the correct amount of liquid. The second stop was to make sure that all of the liquid taken up by the pipettor has left into the desired tube. The final reactions had the same amount of liquid because the micropipettor was very specific as to how much liquid was put into each tube. No liquids were left behind in the tubes where the DNA samples and PCR reactions mixed. Our labeling scheme followed that of the PDF file for the lab; nothing needed to be changed.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone:Samsung Galaxy S6
Flash:No
ISO setting:0
White Balance:0
Exposure:0
Saturation:0
Contrast:0
Camera set-up
Distance between the smart phone cradle and drop = 5cm
Placing Samples onto the Fluorimeter
Place 80 microliters of SYBRI onto the glass slide
Add the specified concentration of the 2X Calf Thymus DNA Solution to the SYBRI.
Align the blob of liquid with the blue LED light so that it is focused to the middle of the black optic fiber on the other side of the blob.
Repeat steps 1-3 for each of the 6 concentrations.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Concentration
Low Concentration
Zero Concentration
Calibrator Mean Values
sample number
image number
final DNA concentration in sypr green 1 solution (micrograms/mL)
AREA
Mean Pixel Value
RAWINTDEN of the drop
RAW INTDEN of the background
5ml
1
2.5
58604
140.108
8210911
480001
5ml
2
2.5
59911
136.987
8207050
446427
5ml
3
2.5
60172
188.288
11028817
420684
2ml
4
1
102423
182.546
18696933
663397
2ml
5
1
90196
194.098
17506839
606940
2ml
6
1
100122
187.709
18793760
669545
1ml
7
0.5
53204
169.577
9022165
560539
1ml
8
0.5
58604
172.399
10103271
597953
1ml
9
0.5
54628
171.86
9388379
551485
.5ml
10
0.25
80526
139.528
11235602
584496
.5ml
11
0.25
83021
136.218
11308946
670847
.5ml
12
0.25
86500
142.208
12300968
587701
.25ml
13
0.125
78723
123.066
968128
604677
.25ml
14
0.125
83330
106.742
8894813
1242142
.25ml
15
0.125
78723
107.274
8444908
1184645
0ml
16
0
85054
83.392
7262906
821217
0ml
17
0
83330
83.744
6978426
783993
0ml
18
0
97972
88.35
8655810
896692
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
"PCR Product Concentration (µg /mL)
(Step 5 calculation)"
Total Dilution
"Initial PCR Product Concentration
(µg /mL)
(Step 6 calculation)"
negative
9363938
13.8588
12
166.3056
1=1
8239334
7.2657
12
87.1884
1=2
7817477
4.792
12
57.504
1=3
5966138
-6.061
12
(-72.732)
2=1
6583928
-2.45
12
(-29.4)
2=2
7824816
4.836
12
58.032
2=3
7038079
0.223
12
2.676
positive
17872824
1006.774
12
12081.288
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control
Positive Control
PCR Results: PCR concentrations solved
PCR product tube label
image number
AREA
Mean Pixels
RAWINTDEN of the drop
RAWINTDEN of the background
RAWINTDEN drop - background
negative n
1
96670
108.587
10497142
877995
9619147
negative n
2
89338
102.517
9158627
720565
8438062
negative n
3
93357
107.004
9989540
880811
9108729
1_1
4
87218
100.252
8743812
666542
8077270
1_1
5
84636
101.662
8600843
669491
7931352
1_1
6
88166
102.453
9032895
631497
8401398
1_2
7
86088
101.123
8705476
720871
7984605
1_2
8
86325
100.495
8675211
718113
7957098
1_2
9
81988
102.383
8394203
743854
7650349
1_3
10
75812
84.763
6426086
599890
5826196
1_3
11
69934
84.077
5879847
558317
5321530
1_3
12
77062
86.93
6698979
592900
6106079
2_1
13
81541
87.041
7097449
768089
6329360
2_1
14
81336
88.105
7166089
807823
6358266
2_1
15
85111
89.231
7594505
756010
6838495
2_2
16
83328
102.712
8558802
755442
7803360
2_2
17
82273
106.212
8738353
614264
8124089
2_2
18
82292
103.345
8504506
658235
7846271
2_3
19
68930
105.298
7258196
390891
6867305
2_3
20
72988
107.921
7876923
482068
7394855
2_3
21
69672
109.464
7626573
417721
7208852
Positive P
22
85083
210.537
17913085
427129
17485956
Positive P
23
84721
214.511
18173620
424455
17749165
Positive P
24
88124
211.802
18664807
405016
18259791
PCR Results: Summary
Our positive control PCR result was 2.5 μg/mL
Our negative control PCR result was 0 μg/mL
Observed results
Patient 79685: The water droplet was all blue when viewing them in the Florimeter and contained no amount of green disease.
Patient 90772: The water droplet was all blue when viewing them in the Florimeter and contained no amount of green disease.
Conclusions
Patient 79685: This patient's results came back negative which means that they did not have the disease.
Patient 90772: This patient's results also came back negative which means that they also did not have the disease.