Name: Walter C. Bregon Role(s): Chief of Neurosurgery
Name: Nathan LeFort
Name: Abdurrahman Darwish
Name: Hau Nguyen
LAB 5 WRITE-UP
Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S5
Flash: Inactivated
ISO setting: 800
White Balance: Auto
Exposure: Highest setting available
Saturation: Highest setting available
Contrast: Lowest setting available
Even though we set these parameters, the Galaxy was sensitive enough to get good calibration results with DNA concentration.
Calibration
1. Turn on Blue LED excitation light.
2. Turn on the camera of the smartphone and adjust the camera setting as those listed above. If your phone won't allow you to change settings than don't worry about this. Just hope that you bought a good enough phone that has high quality picture taking capabilities.
3. Place the phone on the cradle as close to a 90 deg angle as possible from the slide so that it takes a picture of the SYBR GREEN/Calf Thymus DNA solution drop sideways. You may need to raise the fluorimeter using the plastic trays provided in order to make sure the camera is sideways to the drop. This will allow ImageJ to filter the blue light easier.
4. Adjust the distance between the smartphone on its cradle and the first two rows of the slide (where the drop will be placed) so that it is as close as humanly possible without causing the image to blur. This distance is atleast 4cm away from the drop and should be measured with a metal ruler.
5. Record this distance and try not to move the camera, cradle, or fluorimeter because this can change the image due to the difference in light collect between the different lengths.
Distance between the smart phone cradle and drop "picture distance" = 7.3 cm
Solutions Used for Calibration
Initial [2X Calf Thymus DNA Soln] (ug/ml)
Vol of the 2X DNA Soln (uL)
Vol of the SYBR GREEN I Dye soln (uL)
Final [DNA] in SYBR GREEN I soln (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
Photo of fluorimeter set-up
Placing Samples onto the Fluorimeter
Deposit a 80 uL drop of SYBR GREEN I in middle of the 1st 2 rows of the slide using the pipettor so the drop is stationary & round.
Add 80 uLs of the 2X DNA soln.
Align the drop by moving the slide so that the blue LED light goes through the middle of the drop and is not refracted to any side.
Set up the timer on your camera (of your smartphone) to take a picture of the drop after you have closed the light (black) box over the fluorimeter & camera. This box removes as much light as possible so that the SYBR GREEN doesn't start to react before we need it to causing the results to be imprecise.
Take 3 images of the drop while keeping the camera focused and not moving anything.
Remove the box while not moving the phone. If you need to adjust for any accidental movement of the cradle, the phone, or the fluorimeter setup at any time, be sure to use the metal ruler to set the same "picture distance" you used before.
Use the pipettor to remove the 160 uL drop from the surface.
Move the slide to the next position so that the next sample drop will have a clean surface to 'sit' on.
Repeat steps 1-8 for all the other concentrations of Calf Thymus DNA as outlined on the table above.
You can use another slide if you make a complete mess of a slide. I paid for them so you don't have to worry about expense.
Don't forget to do the zero [DNA] solution.
Data Analysis
Representative Images of Negative and Positive Samples
Sample Droplet with no DNA
Sample Droplet with DNA (positive signal
Image J Values for All Calibrator Samples
Table 1: Edited Raw Data
Final DNA concentration in SYBR Green I solution (µg/mL)
Area
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
RAWINTDEN DROP - BACKGROUND
2.5
74904
128.083
9593914
45481
9548433
1
61875
123.303
7620386
91250
7529136
0.5
52292
87.918
4597419
114789
4482630
0.25
43266
59.326
2566780
120847
2445933
0.125
25922
32.018
829979
56835
773144
0
36072
71.246
2570001
239501
2330500
Calibration curve
PCR Results Summary
Our positive control PCR result was 0.6510 μg/mL
Our negative control PCR result was -0.3926 μg/mL
Observed results
Patient 41180 : The image of the drop was green. The 1st, 2nd and 3rd replicates, respectively were: -0.4362, -0.5876, 0.7658
Patient 68784: The image of the drop was almost no green to clear in color. The 1st, 2nd and 3rd replicates, respectively were: -0.5428, -0.3680, -0.3623
Conclusions
Patient 41180 : Patient 1 had inconclusive results possibly due to sample contamination or possibly because of forgetting to put the cyber green in to the sample droplet or allowing the droplet to be exposed to light for longer than it should have, etc. Patient 1 probably was negative for the [DNA] with respect to cyber green in PCR but it turned out to be inconclusive because two of the replicates were negative but one was positive.
Patient 68784 : Patient 2 was negative because all three replicates were negative just like the negative sample. This means that the sample droplet showed very little green if any to clear in color. Again the causes for this are the same as previously written for the patient 1.
SNP Information & Primer Design
Background: About the Disease SNP
SNP stands for "single nucleotide polymorphism". Polymorphisms at specific points in the DNA sequence are what make each individual different from the next. They are what leads to genetic variation between people. SNPs are heavily influential to how an individual looks, acts, if they are susceptible to certain diseases, and even drug efficacy and side-effects.
In this lab, the disease was coronary heart disease in humans. Students looked at the gene SNP rs268 to see if the disease was present. The gene that codes for the disease was present if the polymorphism changed from AAT to AGT, which is the disease-associated allele.
Primer Design and Testing
Non diseased Forward primer (20): 5’-AATCTGGTCTATGAGATCAA
Non diseased Reverse primer (20): 5’-GAAACACCAGGGCTCAGGGT
Diseased Forward primer (20): 5’-AATCTGGGCTATGAGATCAG
Diseased Reverse primer (20): 5’-GAAACACCAGGGCTCAGGGT
In a Non diseased person, when you run the PCR, the reverse prime will duplicate, however, the forward primer will not. Therefore, you are only getting the result for half of the strand. Additionally, the reverse primer will not pair up with the forward primer. In a diseased primer, both forward and reverse primers duplicate, and they will base pair according to their nucleotide sequence.
BME 100 Spring 2015
