Set phone to video mode then simply took three screenshots of to get our pictures
Calibration
Distance between the smart phone cradle and drop = 5cm
Solutions Used for Calibration
Initial Concentration of n2X Calf Thymus DNA solution (micrograms/mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR GREEN I Dye solution (µL)
Final DNA concentration in SYBR Green I solution (µg/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Choose a camera to use, set it to the specifications described in the workbook and place it in the stand.
Set up the Flourimeter.
Place slide in the flourimeter with the smooth side down and the rough side up.
Place one, 80 micro-mL drop of SYBR Green on the slide, between two of the dots on the slide.
Place one, 80 micro-mL drop of a calf thalmus solution on the slide so it combines with the SYBR Green.
Adjust the slide so the light shines through the drops.
Adjust and record camera distance.
Start camera video and close the fluorimeter.
Wait a few seconds and remove camera, take screenshots on the video at different times and send them to be analyzed.
Discard the droplets and slide.
Repeat.
Data Analysis
Representative Images of Negative and Positive Samples
Positive Sample
Negative Sample
Image J Values for All Calibrator Samples
Final DNA Concentration in SYBR Green I Solution (µg/mL)
Area
Mean Pixel Value
RAWINTDEN of the drop
RAWINTDEN of the Background
RAWINTDEN DROP - BACKGROUND
2.5
72592
27.453
1992874
0
1992874
2.5
81840
24.854
2034060
6
2034054
2.5
69124
35.062
2423628
0
2423628
1
100748
32.737
3298170
69
3298101
1
90320
36.084
3259073
6
3259067
1
90392
36.403
3290496
67
3290429
0.5
3578
18.658
66760
1605
65155
0.5
52088
15.451
804792
1540
803252
0.5
62452
13.06
815628
2564
813064
0.25
113076
16.741
1892954
9935
1883019
0.25
68308
26.692
1823278
9604
1813674
0.25
60128
29.933
1799822
8492
1791330
0.125
71016
27.528
1954937
13177
1941760
0.125
52480
35.86
1881914
3204
1878710
0.125
73144
73144
27.193
4377
-4349.807
0
67164
28.526
1915935
4863
1911072
0
78472
21.653
1699177
3126
1696051
0
73296
22.284
1633341
1343
1631998
Final DNA Concentration in SYBR Green I Solution (µg/mL)
1
2
3
Average
Standard Deviation
2.5
3298101
3259067
3290429
3282532.333
20680.45689
1
1992874
2034054
2423628
2150185.333
237701.7397
0.5
1883019
1813674
1791330
1829341
47810.13854
0.25
1911072
1696051
1631998
1746373.667
146184.3099
0.125
1941760
1878710
4349.807
1274939.936
1100814.826
0
65155
803252
813064
560490.3333
429001.0352
Calibration curve
Image J Values for DNA Samples
PCR Product Tube Label
Area
Mean Pixel Value
RAWINTDEN of the drop
RAWINTDEN of the Background
RAWINTDEN DROP - BACKGROUND
P
66936
28.847
193087
3483
189604
P
47080
37.904
1784535
3396
1781139
P
58340
32.315
1885230
8297
1876933
N
36472
34.757
1267652
0
1267652
N
59360
22.418
1330703
0
1330703
N
48700
27.63
1345571
16
1345555
1.1
53964
25.715
1387673
16
1387657
1.1
52332
35.469
1856168
6575
1849593
1.1
56356
32.198
1814544
1574
1812970
1.2
39616
34.895
1382389
0
1382389
1.2
51536
27.49
1416732
0
1416732
1.2
69756
24.391
1457486
0
1457486
1.3
50404
36.576
1843555
273
1843282
1.3
50656
37.629
1906116
6616
1899500
1.3
6287
26.116
164194
1234
162960
2.1
65224
27.275
1778976
70
1778906
2.1
43356
38.869
1685201
2480
1682721
2.1
4526
31.611
143071
212
142859
2.2
65880
37.459
2467789
3149
2464640
2.2
65064
35.556
2313390
683
2312707
2.2
5838
35.754
208730
837
207893
2.3
37080
37.087
1375187
2508
1372679
2.3
42904
33.525
1438374
3256
1435118
2.3
36256
36.624
1327825
400
1327425
PCR Product Tube Label
1
2
3
Average
Standard Deviation
P
189604
1781139
1876933
1282558.667
947737.5956
N
1267652
1330703
1345555
1314636.667
41361.99527
1.1
1387657
1849593
1812970
1683406.667
256780.4705
1.2
1382389
1416732
1457486
1418869
37594.08104
1.3
1843282
1899500
162960
1301914
805689.0535
2.1
1778906
1682721
142859
1201495.333
918066.4754
2.2
2464640
2312707
207893
1661746.667
1261363.854
2.3
1372679
1435118
1327425
1378407.333
54074.54035
Calculating Concentration of PCR Product
PCR Product Tube Label
MEAN RAWINTDEN DROP - BACKGROUND
PCR PRODUCT CONCETRATION
INITIAL PCR PRODUCT CONCENTRATION
P
1282558.667
0.313086477
3.75703772
N
1314636.667
0.348630203
4.183562439
1.1
1683406.667
0.757242338
9.086908057
1.2
1418869
0.464123861
5.569486335
1.3
1301914
0.334532972
4.014395664
2.1
1201495.333
0.223265012
2.679180139
2.2
1661746.667
0.733242179
8.798906142
2.3
1378407.333
0.419290691
5.031488298
PCR Results Summary
Our positive control PCR result was 3.757 μg/mL
Our negative control PCR result was 4.183 μg/mL
Observed results
Patient 81166 : All images taken of patient one's DNA samples seemed to be negative. None of them glowed green. All of this patients concentration results were above the negative control.
Patient 54283 : All images taken of patient two' DNA samples appeared to be negative as well as none of them glowed green like the positive sample did. However, not all of the values of concentration found for patient two were above the negative control.
Conclusions
Patient 81166 : Based on the images alone, this patient tested negative.The data calculated shows all concentrations to be above both the negative and positive controls which suggests the sample to be negative as the negative concentration was found to be higher than the positive. The final conclusion is this patient is negative.
Patient 54283 : Based on the images alone, this patient tested negative. The data calculated shows two of the three concentrations to be above both the negative and positive control and one below. This still impliess that this patient is negative.
Examining only the data collected, it is hard to determine a result as all the data found using Image J is relatively similar. The positive and negative control concentrations were very near to each other with the negative control having a higher concentration than the positive control. The data collected for the patients ranged both above and below the controls.This suggests an error was made and further testing would be needed to get a definitive result.
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is a subunit of a nucleic acid and a polymorphism is when two or more clearly different phenotypes exist within the same population. Therefore, a SNP, or single nucleotide polymorphism, is simply a DNA sequence variation where a single nucleotide is different when compared to others of the population.
The variation rs1699154 is found in Homo Sapiens a.k.a humans. It is located on the chromosome 21:34370656. This SNP is pathogenic and associated with the KCNE2 gene-- which stands for potassium voltage-gated channel-- and the cardiac disease Long QT Syndrome.
Primer Design and Testing
The KCNE2 gene stands for potassium voltage-gated channel Isk-related family, member 2. The non-disease allele is TTC but the disease-associated allele contains the sequence is CTC. The numerical location of this SNP is 34370656. The numerical position needed for the non-disease reverse primer is exactly 200 bases to the right of the SNP at 34370856. the non-disease forward primer is 5'-catggtgatgattggaatgt, the reverse primer sequence is 5'-cccttatcagggggacattt. Similarly, the disease forward primer is 5'-catggtgatgattggaatgc and the disease reverse primer is cccttatcagggggacattt.