Distance between the smart phone cradle and drop = 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 (µg/mL)
5
80
80
2.5
2
80
80
1
1
80
80
.5
.5
80
80
.25
.25
80
80
.125
0
80
80
0
Placing Samples onto the Fluorimeter
First, make sure proper PPE is being used, this includes gloves and a lab coat.
Obtain a micropipet that is able to transport 80 microliters, fluorimeter, glass slides, camera, stand for camera, and box to cover the system.
Set up the system so that the camera will be able to take pictures of the side view of the sample.
Place 80 microliters of the SYBER green on the rough side of the glass slide between the two dots. Then place 80 microliters of the sample being used on the orb of SYBER green.
Carefully place the glass slide in the fluorimeter so the beam of light is passing through the orb, then cover the system with the box no light will be able to penetrate.
Set up camera up to timer of 5 seconds, press the take picture button and close the open end of the box.
Repeat steps 5 through 6 for multiple samples being tested.
Data Analysis
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
DNA concentraion
AREA
Pixel count
drop
background
drop-background
mean
STD
5
39576
101.092
4000822
128151
3872671
2667215
1796151
2
39576
66.07
2614781
161980
2452801
1743198
1120036
1
39576
12.744
504367
78407
425960
336244.7
185107.4
.5
39576
23.49
929623
140840
788783
619748.7
363486.2
.25
39576
19.055
754126
141578
612548
502750.7
261876.5
0
39576
5.143
203558
147809
55749
135705.3
60946.69
Label
AREA
Pixel count
drop
background
drop-background
mean
STD
Product
initial
positive
24056
106.562
2782287
85221
2687066
185485
1244858
3.154
.0262885
negative
26919
10.407
280149
82554
197595
186766
81030.43
-.15849
-0.01321
1-1
28646
68.8
1970856
1919927
1313904
1313904
893300
2.080193
0.173349
1-2
22392
106.919
2394131
95574
2298557
1596087
106740
2.640656
.2200
1-3
17577
101.955
1792056
60166
1731890
1194706
802615.5
1.84344
.15362
2-1
21980
135.48
2977998
84486
2893512
1985332
1344544
3.41376
.28448
2-2
24168
137.49
3322893
94492
3228401
2215262
1500107
3.9704
.3225
2-3
21090
136.205
2872568
81910
2790658
1915045
1296654
3.274159
.272849
Calibration curve
PCR Results Summary
Our positive control PCR result was _3.15___ μg/mL
Our negative control PCR result was __-0.15__ μg/mL
Observed results
Patient 76447 : A value of 2.19μg/mL was obtained for our final product. It was observed to be a green when we placed the green dye in it.
Patient 58040 : A value of 3.55μg/mL was obtained for our final product. It was observed to be a bright green when we placed the green dye into it.
Conclusions
Patient 76447 : The value for this patient is higher than the negative control, but lower than the positive control. This means that it has a concentration of DNA, but that concentration is still lower than our controls concentration. This is why the color is not as bright.
Patient 58040 : The value for this patient is higher than both the negative and the positive control. This shows that it has a higher concentration of DNA than the positive control. This explains the bright green color of the droplet.
SNP Information & Primer Design
Background: About the Disease SNP
Single Nucleotide Polymorphisms which are frequently called snips, are the most commonly attributed genetic variation among humans. SNP's occur normall throughout a humans DNA replacing single nucleotides- the building block of DNA. These snips occur once per 300 nucleotides roughly and can act as bio markers for scientists. These snips are flags for scientits to locate where or which gene is causing a certain disease. SNP's can be used to track inheritance of diseases gnes within blood.
Primer Design and Testing
The reulsts of our primer test were conclusive. The non-disease primers or the unaltered section, the test proved to be successful of a match. When we tested the disease primer the test was a fail which means that the two sections where not a match. This is because of the single replacement of a nucleotide in the diseased primer.