|
OUR TEAM
LAB 5 WRITE-UP
Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone 5s
- Flash: Off
- ISO setting: Auto
- White Balance: Auto
- Exposure: Auto
- Saturation: Auto
- Contrast: Auto
Calibration
The phone cradle should be 4 centimeters from the fluorimeter. The photo should be taken edge-on which means you should see the side of the plate and the drop. The photo needs to be taken with no light so it is suggested that you set a 3 second timer on the phone and turn off the flash. No light should enter the box while the picture is being taken.
- 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 the 2X DNA solution (µL) |
Volume of the SYBR Green 1 Dye solution (µL) |
Final DNA concentration in SYBR Green 1 solution (µg/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
- Use the switch for the Blue LED to activate the excitation light.
- Adjust the settings of your smartphone's camera and make sure the camera is on.
- Adjust the height of the fluorimeter with the plastic trays so your smartphone takes the photos of the drop sideways. Your smartphone should be in the cradle at a right angle from the slide.
- Adjust your smartphone so that is is about 4 centimeters away from the slide, or at close as you can get it to the slides without the image being blurry.
- Use a ruler to record the distance between your smartphone and the drop. Try not to move any of the components too much or else the light collected might show a significant difference between images.
- Place a 80 microliter drop of SYBR GREEN I between the first two rows of the slide. Then add a 80 microliter drop of a calf thymus solution. This is a sample.
- Adjust the slide so that the blue LED light is focused by the drop to the cener of the black fiber optic on the other side of the slide.
- Set the timer on the smartphone so that photos may be taken after covering the fluorimeter and the phone without letting in excess light.
- Make sure the drop is focused and take 3 photos.
- Remove the box without moving the smartphone.
- Take off the 160 microliter drop and readjust the slide to the next position.
- Repeat steps 6-11 for the other solutions of calf thymus DNA.
Data Analysis
Representative Images of Negative and Positive Samples
Error creating thumbnail: File missing
This image represents a sample with no DNA present.
Error creating thumbnail: File missing
This image represents a sample with DNA present.
Image J Values for All Calibrator Samples
| Final DNA concentration
|
Area
|
Mean Pixel Value
|
Raw Intden Drop
|
Raw Intden Background
|
Raw Intden Drop- Background
|
| 0 |
20028 |
53.351 |
1068506 |
50928 |
1017578
|
| 0 |
19320 |
55.489 |
1072042 |
54994 |
1017048
|
| 0 |
21484 |
58.681 |
1260713 |
58809 |
1201904
|
| 0.25 |
25960 |
102.281 |
2655209 |
77962 |
2577247
|
| 0.25 |
28664 |
102.309 |
2932581 |
85834 |
2846747
|
| 0.25 |
30910 |
95.699 |
2958067 |
59798 |
2898269
|
| 0.5 |
26840 |
115.051 |
3087973 |
131143 |
2956830
|
| 0.5 |
29768 |
112.369 |
3344991 |
81490 |
3263501
|
| 0.5 |
26076 |
111.946 |
2919104 |
60412 |
2858692
|
| 1 |
26544 |
140.603 |
3732158 |
103219 |
3628939
|
| 1 |
26504 |
146.017 |
3870039 |
125554 |
3744485
|
| 1 |
30572 |
139.678 |
4270228 |
137375 |
4132853
|
| 2 |
28500 |
188.764 |
5379771 |
69598 |
5310173
|
| 2 |
31372 |
186.659 |
5855870 |
70684 |
5785186
|
| 2 |
29300 |
189.379 |
5548799 |
67173 |
5481626
|
| 5 |
28960 |
231.246 |
6696891 |
70908 |
6625983
|
| 5 |
29034 |
224.763 |
6525315 |
71385 |
6453930
|
| 5 |
28600 |
231.416 |
6618501 |
85853 |
6532648
|
Calibration curve
Error creating thumbnail: File missing
PCR Results Summary
- Our positive control PCR result was 2.931 μg/mL
- Our negative control PCR result was 1.131 μg/mL
Observed results
- Patient 14376 : The images for patient 14376 showed that the drops were very fluorescent. The drops glowed bright green, and appeared opaque. The DNA concentration for the samples was found to be 4.227 μg/mL.
- Patient 62548 : The images for patient 62548 showed slight fluorescence. They did not show any signs of glowing, but the drops had a green hue and were more translucent than transparent. The DNA concentration for the samples was found to be 0.6797 μg/mL.
Conclusions
- Patient 14376 : The patient tests positive for the disease because the DNA concentration of the samples, 4.227 μg/mL, is much higher than the DNA concentration of the positive control 1.131 μg/mL.
Note: Due to labeling errors, it was not possible to determine the patient IDs of the different samples. The most probable ID was assigned to each sample, but this could be inaccurate.
Background: About the Disease SNP
A disease SNP is made up of a nucleotide and a polymorphism and affects a specific allele sequence. A nucleotide is a structural component of DNA/RNA and consists of bases such as adenine, thymine, guanine, and cytosine. A nucleotide is a deoxyribose sugar group bonded with one of the four bases.
A polymorphism is a genetic variation which is a difference in a DNA strand among individuals. Polymorphism is when two or more phenotypes (physical traits) exist in the same population of species. This may show variations in health among the individuals, and if more than one percent of a population is positive for the variation, it is useful for genetic linkage analysis. The mutation is a missense mutation where there is a single nucleotide change in the codon.
Primer Design and Testing
To find the non-disease forward, we counted 19 bases on the top row from the left including the T base. For the reverse primer, we searched for the 34370856 base (200 to the right from position 34370656) and then counted 19 to the left on the bottom row. For the disease forward primer, we swapped the T with C and that resulted in no results coming up in the search.
Non-disease
Error creating thumbnail: File missing
Disease
Error creating thumbnail: File missing
| 
BME 100 Fall 2014
