Calibration
The camera (iPhone 6) was set up horizontally in front of the fluorimeter, and was placed in the cradle after the camera was adjusted according to the settings listed above. After adjustment, the image was checked to make sure that it was not blurry. The fluorimeter was raised up using one stack, in order to make sure the camera captured the best image possible, and the phone was placed 6.5 cm away from the fluorimeter.
Distance between the smart phone cradle and drop = 6.5 cm
Solutions Used for Calibration
Initial Concentration of 2X 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 (μL/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
Using the mircopipette, place 80μL of the SYBR GREEN I onto the slide in between the first two rows, then add 80μL of of one of the concentrations of calf thymus from the table on top of the first drop of SYBR GREEN I so that one large drop is formed
Make sure the drop is alighned so that the blue LED light is shining through the large drop
Set the timer on the camera being used, and place the cover on the lightbox to make sure that the picture is taken with little to no light shining through Make sure to take 3 focused images of the drop
Set the micropipette to 160μL and remove the large drop from the slide, before moving the slide to the next position
Repeat steps 1-4 for the rest of the concentrations from the table
Data Analysis
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
Final [DNA] in SYBR Green I Sol (ug/mL)
Area
Mean Pixel Value
RAWINTDEN of Drop
RAWINTDEN of Background
RAWINTDEN Drop - Background
2.5-1
35996
199.129
7167852
166312
7001540
2.5-2
40180
211.856
8512366
192104
8320262
2.5-3
41729
209.272
8732706
181855
8550851
0.25
0
0.125
0
0
0
.25-3
41590
159.203
6621241
153802
6467439
.25-2
44992
167.481
7535322
22468
7512854
.125-1
40608
108.934
4423611
150574
4273037
.125-2
40608
116.099
4714549
19321
4695228
.125-3
40608
117.204
4759405
18356
4741049
0-1
35264
75.15
2650074
20400
2629674
0-2
34046
84.366
2872310
30032
2842278
0-3
33419
75.27
2515459
26496
2488963
1.1.1
38296
163.748
6270880
101101
6169779
1.1.2
35684
167.31
5970273
138573
5831700
1.1.2
37744
165.82
6258723
237252
6021471
1.2.1
38020
99.863
3796414
107865
3688549
1.2.2
39654
161.47
6402917
269273
6133644
1.2.3
37690
160.225
6038873
109392
5929481
1.3.1
37428
109.65
4103977
281660
3822317
1.3.2
37125
114.666
4256964
114561
4142403
1.3.3
38202
112.909
4314456
149338
4165118
2.1.1
39281
86.217
3386690
149338
3237352
2.1.2
39281
92.082
3617063
188645
3428418
2.1.3
39281
138.829
5453353
21303
5432050
2.2.1
37798
83.975
3174087
151874
3022213
2.2.2
37919
132.678
5031028
54170
4976858
2.2.2
37519
119.076
4467604
138921
4328683
2.3.1
36114
99.117
3579523
175287
3404236
2.3.2
40432
107.872
4361494
34774
4326720
2.3.3
44097
159.975
7183976
183930
7000046
N-1
40338
94.068
3794510
173768
3620742
N-2
47908
199.848
9574297
219253
9354774
N-3
48908
201.997
9715635
209413
9506222
P-1
51537
209.389
10791274
15784
10775490
P-2
40284
118.864
4788337
148864
4639473
P-3
42216
128.918
5442418
16555
5425863
Calibration curve
PCR Results Summary
Our positive control PCR result was 1.206 μg/mL
Our negative control PCR result was 5.791 μg/mL
Observed results
Patient 21533: The ImageJ green analysis showed a light grey/white streak through the center of the drop, meaning DNA was present, giving a positive result.
Patient 45821 : The ImageJ analysis had no light spot, and matched the calibration test with no DNA, making this patient negative
Conclusions
Patient 21533 : Since the ImageJ green analysis contained the light grey/white streak in the center, DNA was present, so it matched the positive control.
Patient 45821 : There was no light spot on the Image J analysis for this patient, so it matched the negative control sample.
SNP Information & Primer Design
Background: About the Disease SNP
What is a nucleotide? The organic base units of DNA and RNA
What is a polymorphism? A point mutation that is expressed in multiple forms within a population.
What species is this variation found in? (latin name) Homo Sapien
What chromosome is the variation located on? 8
What is listed as the Clinical significance of this SNP? It is contained within a pathogenic allele
Which gene(s) is this SNP associated with? LPL
Click the PubMed link to view summaries of research associated with the SNP. What disease is linked to this SNP? Coronary heart disease
What does LPL stand for? Lipoprotein Lipase
What is the function of LPL? To find out, click the LPL link. Look for “Gene ontology” in the right hand list and click it. Write the first three unique terms you see...
apolipoprotein binding
heparin binding
lipoprotein lipase activity
What is an allele? A variation in a gene
The disease-associated allele contains what sequence? aat
The numerical position of the SNP is 19956018
Non-disease forward primer (20 nt): aatctgggctatgagatcaa
The numerical position exactly 200 bases to the right of the disease SNP is: 19956218
Non-disease reverse primer (20 nt): gaaacaccagggctcagggt
Disease forward primer (20 nt): aatctgggctatgagatcag
Disease reverse primer (20 nt): gaaacaccagggctcagggt
Primer Design and Testing