Distance between the smart phone cradle and drop = 8.7cm
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)
5
80
80
2
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0.0
80
80
0.0
Placing Samples onto the Fluorimeter
Put on your lab coat and a pair of gloves.
Set up camera and slide. Make sure that the camera is at least 4 cm away from the slide.
Pick up the micropipette and set it to 80 microliters.
Put a new tip on the micropipette.
Put the micropipette into the cyber green and collect 80 microliters.
Put the cyber green onto the slide using the first two open spaces in the middle of the slide.
Eject tip from micropipette and put a new one on.
Put the micropipette inside of the positive control and take 80 microliters.
Place the positive control inside of the cyber green that is currently on the slide.
Turn on the blue light.
Take 3 pictures of the mixture.
Repeat these steps for the next 5 mixtures.
Data Analysis
Representative Images of Negative and Positive Samples
Negative Sample
Positive 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
331364
198.732
65852597
17649621
48202976
2.5
342228
199.351
68223421
16724921
51498500
2.5
250184
199.324
49867751
1240020
48627731
1
296560
198.506
58868847
10437897
48430950
1
281220
197.943
55665421
10138481
45526940
1
298592
182.37
54454217
9172072
45282145
0.5
348032
174.117
60598450
10459380
50139070
0.5
440128
173.75
76472163
12152830
64319333
0.5
528596
58.82
31092099
1129060
29963039
0.25
464696
145.554
67638160
11353152
56285008
0.25
718056
52.967
38032999
1626013
36406986
0.25
686180
150.246
103096002
19177502
83918500
0.125
680324
56.656
38544379
6472829
32071550
0.125
504300
124.562
62816687
21802388
41014299
0.125
516896
124.628
64419793
22990441
41429352
0
611476
38.406
23484622
4934774
18549848
0
524308
109.757
57546518
21610863
35935655
0
647168
108.69
70340542
27210814
43129728
Final DNA Concentration in SYBR Green I Solution (µg/mL)
R1
R2
R3
RAWINTDEN Mean
Standard Deviation
2.5
48202976
51498500
48627731
49443069
1792680.019
1
48430950
45526940
45282145
46413345
1751578.888
0.5
50139070
64319333
29963039
48140480.67
17265123.92
0.25
56285008
36406986
83918500
58870164.67
23861019.82
0.125
32071550
41014299
41429352
38171733.67
5286988.54
0
18549848
35935655
43129728
32538410.33
12637190.3
Calibration curve
PCR Results Summary
PCR Dilutions:
PCR Tube Label
Volume of Diluted PCR Product Solution (μL)
Volume of SYBR Green I Dye Solution (μL)
Dilution 1
Dilution 2
Total Dilution (simplified fraction)
G4 +
80
80
(1/6)
0.5
(1/12)
G4 -
80
80
(1/6)
0.5
(1/12)
G4 1-1
80
80
(1/6)
0.5
(1/12)
G4 1-2
80
80
(1/6)
0.5
(1/12)
G4 1-3
80
80
(1/6)
0.5
(1/12)
G4 2-1
80
80
(1/6)
0.5
(1/12)
G4 2-2
80
80
(1/6)
0.5
(1/12)
G4 2-3
80
80
(1/6)
0.5
(1/12)
PCR ImageJ Chart:
PCR Tube Label
Area
Mean Pixel Value
RAWINTDEN of the drop
RAWINTDEN of the background
RAWINTDEN Drop - BACKGROUND
G4 +
238260
206.528
49207455
8102221
41105234
G4 +
475036
82.259
39075423
2140855
36934568
G4 +
614748
81.835
50307831
2596113
47711718
G4 -
453896
166.741
75683044
15204655
60478389
G4 -
389668
70.07
27303846
1608565
25695281
G4 -
453300
67.805
30735807
1856974
28878833
G4 1-1
461720
66.512
30710099
1664280
29045819
G4 1-1
501084
69.275
34712669
1923263
32789406
G4 1-1
407032
185.24
75398569
15819040
59579529
G4 1-2
382272
185.781
71018867
14446760
56572107
G4 1-2
379832
185.004
70270329
12930916
57339413
G4 1-2
546620
77.236
42218650
2009507
40209143
G4 1-3
424532
171.574
72838811
13651183
59187628
G4 1-3
350160
169.812
59461422
1054940
58406482
G4 1-3
636212
79.321
50465104
2529074
47936030
G4 2-1
342228
176.669
60461158
8916351
51544807
G4 2-1
352960
193.364
68249727
10442955
57806772
G4 2-1
477744
195.703
93495795
9042113
84453682
G4 2-2
327048
192.788
63050943
9830362
53220581
G4 2-2
627364
192.629
120848790
16995345
103853445
G4 2-2
513836
193.179
99262128
13887770
85374358
G4 2-3
305496
155.534
47514967
7818637
39696330
G4 2-3
257640
66.847
17222497
772354
16450143
G4 2-3
234740
183.909
43170690
7086446
36084244
PCR DNA Calculations:
PCR Tube Label
RAWINTDEN Mean
PCR Product Concentration (μg/mL)
Initial PCR Product Concentration (μg/mL)
G4 +
41917173.33
0.639057778
0.053254815
G4 -
38350834.33
-0.549721889
-0.045810157
G4 1-1
40471584.67
0.157194889
0.013099574
G4 1-2
51373554.33
3.791184778
0.315932065
G4 1-3
55176713.33
5.058904444
0.42157537
G4 2-1
64601753.67
8.200584556
0.683382046
G4 2-2
80816128
13.605376
1.133781333
G4 2-3
30743572.33
-3.085475889
-0.257122991
Our positive control PCR result was 0.053254815 μg/mL
Our negative control PCR result was -0.045810157 μg/mL
Observed results
Patient 16819 :
The droplets for patient 16819 had a big green area of DNA in the middle of the drops. DNA was not found around the top or bottom of the drop. Small amounts of green touched the sides of the droplet. Based on the calculation the amount of DNA for patient 16819 is 0.250202 μg/mL.
Patient 59134 :
The droplets for patient 59134 had more green in comparison to those of patient 16819. The green concentrated in the middle, touched the tops of the droplets, more densely touched the sides of the droplets, and still did not touch the bottom. Based on the calculation the amount of DNA for patient 59314 is 0.520013.
Conclusions
Patient 16819 :
After a comparison of the values of positive and negative controls compared to the values of patient 16819, it was determined that the values fell closer to the positive control. For this reason patient 16819 was determined to be positive for SNP.
Patient 59134 :
After a comparison between the values of the positive and negative controls to the values of patient 59134, it was determined that the patient's values fell closer to the positive control. Because of this, patient 59134 was determined to be positive for SNP.
Error Analysis:
Errors were known to have occurred because some of the values obtained are impossible (negative values). The software was used correctly with accordance with the procedure explained in the Lab Workbook. After examination, some of the error can be attributed to different sized images of the droplets because they were taken with different zooms, causing error in the ImageJ calculations. The camera was alinged manually to the droplet each time, but zooming was necessary, and a standard zoom was not determined at the time of taking the pictures.
SNP Information & Primer Design
Background: About the Disease SNP
Part 1:
What is a Nucleotide?
A nucleotide is the primary building block of nucleic acids. They are composed of a nitrogenous base, a five-carbon sugar, and a phosphate group.
What is a Polymorphism?
A polymorphism is when two different phenotypes exists in the same population. For example, there are cheetahs who display a spotted fur, and there are cheetahs who display black fur.
rs268
This variation is found in homo sapiens.
The variation is located on chromosome 8
The clinical significance of this SNP is that it is pathogenic.
This SNP is associated with the LPL gene
A disease associated with this SNP is Coronary Heart Disease
Part 2:
LPL stands for Lipoprotein Lipase
The LPL has several functions:
apolipoprotein binding
heparin binding
lipoprotein lipase activity
An allele is a varying form of a gene.
The disease-associated allele contains the sequence: AGT
The numerical position of the SNP is: 19956018
Primer Design and Testing
When run through the primer test, the non-diseased primer was found to be in the human genome. This is because it is normally produced in the body. Conversely, the diseased primer returned no match because it does not appear in a healthy human genome, as it is a mutation.
The non-disease forward primer (20 nt): 5’ AATCTGGGCTATGAGATCAA
The numerical position exactly 200 bases to the right of the disease SNP is: 19956218
The non-disease reverse primer (20 nt): 5’ TGGGACTCGGGACCACAAAG
Disease forward primer (20 nt): 5’ AATCTGGGCTATGAGATCAG
Disease reverse primer (20 nt): 5’ TGGGACTCGGGACCACAAAG