1. Place fluorimeter on the table and turn on the blue LED by flipping the switch
2. Place the slide (smooth side down) in the fluorimeter
3. Set timer on camera to 3-5 seconds and insert into cradle a fixed distance from fluorimeter.
4. Adjust height of fluorimeter using plastic trays to get an edge-on view through the camera.
Distance between the smart phone cradle and drop = 8cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (ug/mL)
Volume of 2X DNA Solution (uL)
Volume of SYBR GREEN I Dye Solution (uL)
Final DNA Concentration in SYBR Green I Solution (ug/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
.0125
0
80
80
0
Placing Samples onto the Fluorimeter
Using gloves, find the smooth side of the sample plate
Slide the sample plate, smooth side down, into the fluorimeter and turn blue LED light on
Using proper micropipetting technique, draw and place 80 uL of SYBR Green I on the first 2 circles on the slide
Insert new pipette tip and draw and place 80 uL of sample/calibration solution on top of the SYBR Green I drop
Adjust the slide so light illuminates the center of the drop
Data Analysis
Representative Images of Negative and Positive Samples
Sample with no DNA
Sample with DNA
Image J Values for All Calibrator Samples
Final DNA concentration in SYBR Green I solution (ug/mL)
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
RAWINTDEN DROP - BACKGROUND
0
28984
11.886
344498
0
344498
0
61956
6.148
380930
0
380930
0
52800
7.303
385613
0
385613
0.25
122221
13.835
1690956
0
1690956
0.25
41840
17.874
747835
111
747724
0.25
38107
17.224
656373
98
656275
0.5
48064
43.756
2103070
1703
2101367
0.5
47681
70.084
3341658
0
3341658
0.5
47017
69.83
3283185
0
3283185
1
47058
56.929
2678965
222
2678743
1
45932
116.4
5346463
4329
5342134
1
51697
123.216
6369878
1362
6368516
2
49277
104.192
5134266
0
5134266
2
47508
174.654
8297468
3489
8293979
2
42232
182.475
7706288
3118
7703170
5
71196
156.683
11155235
10725
11144510
5
45304
215.618
9768366
2898
9765468
5
55848
182.673
10201907
1658
10200249
Calibration curve
PCR Results Summary
PCR Product Tube Label
MEAN RAWINTDEN DROP-BACKGROUND
PCR PRODUCT CONCENTRATION ug/mL
INITIAL PCR CONCENTRATION ug/mL
34 P
3206761.667
0.603380833
7.24057
34 N
771576.3333
-0.614211833
-7.370542
34 +1-1
651603
-0.6741985
-8.090382
34 +1-2
1011260
-0.49437
-5.93244
34 +1-3
1319958.667
-0.340020667
-4.080248
34 -1-1
6481576
2.240788
26.889456
34 -1-2
4780935.333
1.390467667
16.685612
34 -1-3
2600978.333
0.300489167
3.60587
Our positive control PCR result was 7.24057 μg/mL
Our negative control PCR result was -7.370542 μg/mL
Observed results
Patient 88697 : All the images for patient 1 were dark. There was no identifiable green fluorescence coming from the drop. Patient 1 had an average initial PCR product concentration of approximately -6.034 ug/ml
Patient 48246 : All the images for patient 2 had a clear and indentifiable green fluorescence coming from the drop. Patient 2 had an average initial PCR product concentration of approximately 15.727 ug/mL
Conclusions
Patient 88697 : Patient 1 had an average initial PCR product concentration of approximately -6.034 ug/mL. This value differed from the negative control by approximately 1.337 ug/mL and differed from the positive control by approximately 13.275. Since the concentration is closer to the negative control, patient 1 is negative. All 3 of patient 1's initial concentrations were much closer to the negative control.
Patient 48246 : Patient 2 had an average initial PCR product concentration of approximately 15.727 ug/mL. This value differed from the negative control by approximately 23.098 ug/mL and differed from the positive control by approximately 8.486 ug/mL. Since the concentration is closer to the positive control, patient 2 is positive. All 3 of patient 2's initial concentrations were much closer to the positive control.
SNP Information & Primer Design
Background: About the Disease SNP
Nucleotides are the building blocks of DNA. They consist of Adenine, Thymine, Guanine, and Cytosine. A polymorphism is a presence of genetic variation within a population. In this section we are focusing on SNPs, or single nucleotide polymorphism. SNP rs16991654 is found in homosapiens and is located in the 21:34370656 chromosome. This SNP has a clinical significance because it is pathogenic with diseases like congenital long QT syndromes being linked to it. KCNE2 stands for potassium voltage-gated channels and its function primarily consists of regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction and cell volume.
Primer Design and Testing
To design primer pairs for PCR, the non-disease forward primer contains 20 bases and ends with the disease codon. The reverse non-disease primer also contains 20 bases, and is found 200 base pairs to the right on the bottom strand. To create disease primer pairs, the SNP is applied to the appropriate nucleotide in the forward primer. The reverse primer does not change when diseased. When testing both primers, we found that the search returned the non-diseased sequence since it is part of the healthy human genome. The reason why the diseased variation did not come up in the search is because it is not a part of the healthy human genome, it is a SNP.
Non-disease forward and non-disease reverse primer search
BME 100 Fall 2014
