To set up the camera, we placed it in the phone cradle and set up a timer for three seconds. We placed the phone more than four centimeters away from the fluorimeter and made sure it was even with the drop.
Distance between the smart phone cradle and drop = 4 centimeters
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL)
Volume of 2X DNA Solution (µL)
Volume of 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
Solutions Used for PCR Samples
PCR Product Tube Label
Volume of the Diluted PCR Product solution(μL)
Volume of SYBR Green I Dye Solution (µL)
Dilution 1
Dilution 2
Total Dilution Simplified Fraction
14 1-1
80
80
0.166
.5
0.0833
14 1-2
80
80
0.166
.5
0.0833
14 1-3
80
80
0.166
.5
0.0833
14 Pos.
80
80
0.166
.5
0.0833
14 2-1
80
80
0.166
.5
0.0833
14 2-2
80
80
0.166
.5
0.0833
14 2-3
80
80
0.166
.5
0.0833
14 Neg.
80
80
0.166
.5
0.0833
Placing Samples onto the Fluorimeter
Step One: With the fluorimeter on, put the smooth-side of the slide down into the fluorimeter.
Step Two: Open the camera application and set a three second timer with no flash.
Step Three: Place 80 μL of SYBR Green in between the first two circles in the middle row of the slide.
Step Four: Add 80 μL of the Sample Solution to the SYBR Green on the slide using the micropipette.
Step Five: Focus the fluorimeter light on the center of the drop on the slide.
Step Six: Place the camera approximately 4 cm away from the fluorimeter.
Step Seven: Use the box to cover the fluorimeter and the phone.
Step Eight: Make sure the drop is focused on the camera, press the camera button and lower the flap of the box.
Step Nine: Wait approximately 5 seconds to ensure that the picture is taken without disturbance from outside light.
Step Ten: Reopen the flap and take another two pictures.
Step Eleven: Open the flap and remove the 160 μL drop of SYBR Green and Sample Solution from the slide using the micropipette and discard in the waste container.
Step Twelve: Adjust the slide so the light focuses on the next set of circles.
Steps Thirteen: Repeat steps Two through Eleven until all solutions have been placed on the fluorimeter and photographed.
Data Analysis
Representative Images of Negative and Positive Samples This is the positive sample in the flourimeter.
This is the negative sample in the flourimeter.
Image J Values for All Calibrator Samples
Table 1 Description: Table 1 shows all the data taken during the lab and all of the date extracted from ImageJ.
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
2.5 #1
14176
105.51
1495714
21398
1474316
2.5 #2
14590
99.548
1452403
25555
1426848
2.5 #3
15092
101.881
1537586
28563
1509023
1 #1
15402
86.36
1330121
26902
1303219
1 #2
15272
81.5
1244661
29992
1214669
1 #3
15200
82.865
1259552
27209
1232343
.5 #1
11770
91.15
1072830
16946
1055884
.5 #2
10309
95.702
986588
17298
969290
.5 #3
10748
101.886
1094851
15828
1079023
.25 #1
11398
83.283
949255
23992
925263
.25 #2
10646
79.226
843438
16114
827324
.25 #3
10501
82.391
865190
14462
850728
.125 #1
14750
59.167
872719
26834
845885
.125 #2
13853
60.16
833390
30909
802481
.125 #3
13488
63.964
862752
28086
834666
H2O #1
15668
36.41
570466
29545
540921
H2O #2
16158
37.729
609629
32081
577548
H20 #3
16926
36.272
613933
31559
582374
tube 1-1 #1
14774
98.509
1455374
28109
1427265
tube 1-1 #2
15380
96.776
1488419
25996
1462423
tube 1-1 #3
14520
98.487
1430028
21638
1408390
tube 1-2 #1
12100
107.7
1303256
24759
1278497
tube 1-2 #2
12697
105.207
1335808
22155
1313653
tube 1-2 #3
13792
100.782
1389992
26877
1363115
tube 1-3 #1
12092
85.422
1032918
25923
1006995
tube 1-3 #2
10888
93.493
1017951
20411
997540
tube 1-3 #3
12052
86.662
1044452
23297
1021155
positive #1
16484
87.126
1436182
31255
1404927
positive #2
16390
90.677
1486197
29471
1456726
positive #3
16723
89.235
1492281
32215
1460066
tube 2-1 #1
14916
41.782
623226
28122
595104
tube 2-1 #2
15817
39.806
629618
30172
599446
tube 2-1 #3
15036
41.319
621275
27440
593835
tube 2-2 #1
13192
14.357
189394
26457
162937
tube 2-2 #2
12648
14.322
181147
24540
156607
tube 2-2 #3
12842
15.617
200552
24212
176340
tube 2-3 #1
14256
27.649
394162
28295
365867
tube 2-3 #2
14774
22.203
328032
28679
299353
tube 2-3 #3
14520
21.113
306566
25369
281197
negative #1
16308
42.543
693793
33092
660701
negative #2
16353
45.996
752173
35035
717138
negative #3
15913
45.021
716418
32452
683966
Table 2 Description: Table 2 includes the Mean and Standard Deviation calculations of the Final DNA Concentrations. The Mean calculations and Concentrations were used to create the Calibration Curve.
Final DNA Concentration in SYBR Green I Solution (ug/mL)
Mean
Standard Deviation
0
566947.7
22668.54875
0.125
826677.3
21353.80295
0.25
867771.7
51145.64361
0.5
1031399
63510.8986
1
1250077
46863.06319
2.5
1470062
41252.30801
Table 3 Description: The data from Table 3 were calculated using the Calibration Curve to find the unknown concentrations of SYBR Green Solution. This information was then used to determine if the patients were positive for the gene.
Final DNA concentration in SYBR Green I Solution (ug/mL)
PCR Product Concentration (ug/mL)
Initial PCR Product Concentration (ug/mL)
tube 1-1
1.231454625
14.7774555
tube 1-2
1.044726016
12.53671219
tube 1-3
0.538390938
6.460691251
tube 2-1
-0.135563207
-1.62675848
tube 2-2
-0.839582349
-10.07498819
tube 2-3
-0.594179169
-7.130150025
positive
1.244331765
14.93198118
negative
0.013367366
0.160408391
Calibration Curve
Calibration Curve Description: The calibration curve show the relationship between concentration of the solution and the Mean RAWINTDEN drop background of known concentrations. The concentration data from 2.5 μg/mL was omitted from the line of best fit because it was an outlier.
PCR Results Summary
Our positive control PCR result was 14.93198118 μg/mL
Our negative control PCR result was 0.160408391 μg/mL
Observed results
Patient 1-22157 : The images were more florescent than patient 49284. They had a very strong green color.
Patient 2-49384 : The images had more of a darker color. There was no florescence in the images from patient 49284.
Conclusions
Patient 1-22157 : The SYBR Green fluoresces very well in the presence of the dsDNA.
Patient 2-49384 : There was no florescence in the drop because there was no dsDNA present.
SNP Information & Primer Design
Background: About the Disease SNP
Single Nucleotide Polymorphism (also known as SNP) is a variation within the DNA sequence. This variation is common within the population, meaning it is prevalent in 1% of the population. SNPs are differences in only one DNA nucleotide. Nucleotides are the building blocks for DNA and are made up of a nitrogenous base, sugar, and phosphate. SNPs are typically found in non-coding regions of DNA leading to no visible results of this variation. While most SNPs have no effect on a person's health, they can in certain cases predict disease, response to drugs, and other clinical conditions.
Primer Design and Testing
When testing the DNA sequence of the rs16991654 gene, we looked at both the diseased and non-diseased primers. The forward primer, was the DNA sequence of the top strand read from left to right while the reverse primer was the DNA sequence of the lower strand of DNA read from right to left. When we tested the primers for the non-diseased DNA we found that it returned the sequence of the normal human DNA. When we tested the diseased DNA, no matches were returned. This is because the variation in this diseased DNA is not meant to be in a healthy genome; its a SNP.
Sequence used for Forward Primer. To create diseased primer, the Thymine at the end was changed into a Cytosine.
Sequence used for Reverse Primer. Sequence is to be read from right to left.
BME 100 Fall 2014
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