Distance between the smart phone cradle and drop = 10 cm
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 I Solution (μL)
Final Concentration of 2X Calf Thymus DNA Solution (μg/mL)
0
80
80
0
0.25
80
80
0.125
0.5
80
80
0.25
1
80
80
0.5
2
80
80
1
5
80
80
2.5
Placing Samples onto the Fluorimeter
Put on gloves. Then find the smooth side of the slide.
Retrieve a fluorimeter from the TA and take it to your group's table.
Put the slide from before in its place on the fluorimeter, making sure the smooth side is facing down.
Open the camera app, turn on the timer for 3-5 seconds, and put it on the craddle.
Make sure the camera has a good view of the slide by adjusting either the height of the fluorimeter or the camera.
Using a micropipette, take 80 micro liters of SYBR Green I solution and carefully put it between the front two clear circles of the slide.
Now put 80 micro liters of the sample/calibration over the SYBR Green I drop.
Make sure the that the light is hitting the drop in the middle and goes through the other side by adjust the slide.
The camera should be at closest 4 centimeters and focused. Record how far the camera is from the fluorimeter.
Cover the fluorimeter with the lightbox with one of the flaps up.
Double check that the camera is focused on the drop.
Start the camera's timer and take the last flap down.
Once the picture is taken, dispose of the 160 micro liter drop.
Move on to the next position on the slide.
Repeat all the steps for each sample.
Data Analysis
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
Sample
Area
Mean
Min
Max
IntDen
H2O
122472
22.478
0
255
2752947
0.25
334592
33.476
0
255
11200689
0.5
193988
44.834
0
255
8697194
1
117636
55.702
0
255
6552507
2
189104
71.432
0
255
13506310
5
147412
73.233
0
255
10795378
Negative
98980
70.295
0
255
6957848
Positive
84628
73.603
0
255
6228875
Patient2A
73900
37.997
0
255
2807950
Patient2B
76620
41.83
0
255
3205014
Patient2C
184944
84.546
0
255
15636268
Patient1A
183223
61.598
0
255
11286081
Patient1B
107880
39.85
0
255
4299046
Patient1C
90812
65.6
0
255
5957275
Calibration curve
PCR Results Summary
Our positive control PCR result was 80 μg/mL
Our negative control PCR result was 80 μg/mL
Observed results
Patient 1 (ID: 62525): The one sample with fluorescence seemed similar to the 2 μg/mL sample
Patient 2 (ID: 86787): There was a slight observance of SYBR Green which seemed to range from nothing to .5
Conclusions
Patient 1 (ID: 62525) : This patient possibly has SNP. The first two samples were cracked and the third appeared much more fluorescent so perhaps the amount of sample was not enough for detection when diluted.
Patient 2 (ID: 86787) : This patient does not have SNP.
SNP Information & Primer Design
Background: About the Disease SNP
The Single Nucleotide Polymorphism (SNP) is a DNA sequence variation. One of the known SNPs is rs268 in the lipoprotein lipase (LPL) gene. rs268 is found in a species called Germline, which is a series of germ cells. This variation is located in chromosome 8: 19956018. It is considered as a pathogenic SNP and is associated with Coronary Heart disease.
Primer Design and Testing
After testing the Non-disease primer, both forward and backward, we got successful results assuming calculating it with 50mM salt and 50nM annealing oligo concentration. However, the disease primer testing was not successful where there was " no match found."
BME 100 Spring 2015
