Distance between the smart phone cradle and drop = 7.8 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL)
Volume of the 2X DNA Solution (uL)
Volume of the SYBR Green I 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
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Slide the glass slide onto the fluorimeter with the smooth side down and adjust the glass so that the blue light is shining in between two white circles.
Set up the phone camera to the three second timer and turn the flash off. (If the flash is not able to be disabled, skip this step)
Place the phone in the holder and make sure the glass slide is level with the camera and in focus.
Measure the distance between the phone and the fluorimeter (Make sure the camera is more than 4 cm away, in our experiment we used 7.8cm)
Place 80 uL drops of SYBR Green I solution in the between the two circles that the blue light is illuminating. (Remember to use a new micropipette tip for each different solution and discarding the tips in the red disposable cup.)
Place 80 uL drops of the sample solution onto the SYBR Green I drop that is already on the glass slide.
Place the box over the fluorimeter with the opening on the side of the camera.
Focus the image and click on the timer before covering the box with the lid OR cover the box as much as possible while clicking the shutter manually.
Using the micropipette, remove all 160 uL solution from the slide and discard the solution into the red disposable cup.
Move the slide so the blue light shines between two new white circles.
Repeat this procedure for each sample three times.
Put the glass slide in the sharps container once all the possible white circle have been used.
Bonus: Images of Setup
Above is an image of the basic setup without the black box over it. Below is an image of the setup with the black box over it but without closing the final side so that we could take the picture.
Data Analysis
Representative Images of Negative and Positive Samples
Negative SamplePositive Sample
Image J Values for All Calibrator Samples
PCR Product Label Tube
Area
Mean Pixel Value
RAWINTDEN of Drop
RAWINTDEN of Background
RAWINTEDEN (Drop-Background)
5
498
238.514
118780
755
118025
5
498
237.96
118504
1135
117369
5
498
253.303
126145
1918
124227
2
498
242.853
120941
750
120191
2
498
201.57
100382
659
99723
1
498
170.665
84991
687
84304
1
498
163.55
81448
1206
80242
1
498
171.643
85478
786
84692
0.5
498
115.305
57422
740
56682
0.5
500
113.478
56739
840
55899
0.5
484
113.291
54833
799
54034
0.25
484
68.285
33050
632
32418
0.25
484
76.911
34321
722
33599
0.25
484
70.155
33955
1465
32490
0
484
7.395
3579
982
2597
0
484
7.795
3773
693
3080
0
484
7.905
3826
565
3261
Image J Data
PCR Product Label Tube
RAWINTEDEN (Drop-Background) AVERAGE
Standard Deviation
2.5
119873.67
3784.34
1
109957.00
14473.06
0.5
83079.33
2464.85
0.25
55538.33
1360.34
0.125
32835.67
662.05
0
2979.33
343.26
Calibration curve
The last two values plateau in a non-linear way with the graph above throwing off the line of best fit which is why we excluded them on the graph below.
PCR Results Summary
PCR Product Label Tube
Average
PCR Production Concentration
Initial PCR Production Concentration
G1-1
3964
-0.0349
-0.0029
G1-2
2602.5
-0.0436
-0.0036
G1-3
1945.5
-0.0478
-0.0040
G2-3
5176
-0.0271
-0.0023
G2-2
4825
-0.0294
-0.0024
G2-3
3850
-0.0356
-0.0030
Negative
82357.5
0.4666
0.0389
Positive
122055
0.7206
0.0600
Our positive control PCR result was 0.0600 μg/mL
Our negative control PCR result was 0.0389 μg/mL
Observed results
Patient 85470 : The values on this patient were -.0029, -.0036, and .0040 μg/mL. The drops were not green at all so it was negative.
Patient 90113 : The values on this patient were -.0023, -.0024, and .0030 μg/mL. The drops were not green at all so it was also negative.
Conclusions
Patient 85470 : The results suggest that Patient 85470 is closer to the negative value.
Patient 90113 : The results suggest that Patient 90113 is closer to the negative value.
SNP Information & Primer Design
Background: About the Disease SNP
SNP is the abbreviation meaning Single Nucleotide Polymorphism and affect appearance, respond to drugs, and pre-disposition to diseases. In this lab, the particular SNP that is being analyzed is rs16991654, which is called Homo Sapiens (humans) in latin. On the National Center for Biotechnology (NCBI) page, numerous information on this SNP's can be found including the clinical significance, which is pathogenic or the disease linked to this SNP, which is Long QT syndrome (LQTS). The area of information that we focused on for the majority of the time is the gene sequence, which is KCNE2 where we analyzed the numerical position of the SNP (34370656) and had to not the non-disease forward primer, non-disease reverse primer, the disease forward primer, and the disease reverse primer. KCNE2 is gene coding which regulates numerous functions such as the neurotransmitter release, the heart rate, insulin secretion, neuronal excitability, and several others.
Latin Name
Homo Sapien (Humans)
Chromosome
21:34370656
Clinical Significance
Pathognic
Associated Gene
KCNE2 (Potassium Voltage-Gated Chanel, Isk-related family member)
Associated Disease
Long QT Syndrome (LQTS)
Numerical Postion
34370656
Numerical Position (200 bases to the right)
34370856
Non-Disease Forward Primer
CAT-GGT-GAT-GAT-TGG-AAT-GT
Non-Disease Reverse Primer
CCC-TTA-TCA-GGG-GGA-CAT-TT
Disease Forward Primer
CAT-GGT-GAT-GAT-TGG-AAT-GA
Disease Reverse Primer
CCC-TTA-TCA-GGG-GGA-CAT-TT
Primer Design and Testing
The image below shows the UCSC In-Silico PCR website results for the non-disease forward primer and non-disease reverse primer.
The image below shows the UCSC In-Silico PCR website results for the disease specific primers. The results show that there is "no matches."