While pipetting, our group ran into no problems, it took a couple trials to get the hang of things but after that everything went smoothly. The pre-lab reading gave a great overview of what to expect. We were able to understand the difference between the first and second stop soon after getting the pipettor. As far as the final reactions, we had the same amount of liquid for each product. Also, we extracted all the liquid from the tubes so there was no liquid left in the DNA samples or the PCR reaction mix. Lastly, we were able to keep our labeling scheme the same as last the lab.
Fluorimeter Procedure
Imaging set-up
Samsung S8
Go to camera menu
Deactivate the flash
Set ISO to 800
Set White Balance to Auto
Set exposure to highest setting
Set Saturation to Highest setting
Set contrast to the lowest setting
Set Timer on phone
Have camera on three shot mode
Slide and Phone Position setup
Set phone base about 6 cm away from slide
Make sure the image is at a 90 degree angle view
Slide must be level with camera lens
After starting camera timer, cover with given box to reduce light exposure
Placing Samples onto the Fluorimeter
First, as a control, use the micropipette to place 160 microliters of water onto the slide
Use smartphone to capture three images of water droplet
Use micropipette to extract the water from the slide
Use micropipette to distribute 80 microliters of SYBR GREEN 1 solution onto the slide
Add an additional 80 microliters of the 0.25 concentration Calf Thymus DNA Solution onto the slide
Use smartphone to capture three images of droplet
Extract liquid from the slide using micropipette
Repeat steps 3 through 7, except instead of adding the 0.25 concentration Calf Thymus DNA solution, add the solution of interest (0.5, 1, 2, 5, Positive Control, Negative Control, 1-1, 1-2, 1-3, 2-1, 2-2, 2-3)
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Zero Calf Thymus DNA
Low Calf Thymus DNA
High Calf Thymus DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution
Final DNA concentration in SYBR Green I solution (μg/mL)
Sample Number
RAWINTDEN DROP - BACKGROUND
RAWINTDEN DROP - BACKGROUND
RAWINTDEN DROP - BACKGROUND
Mean
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
3850171
3733338
3558373
3713960.667
146860.9172
2
1
C-2
4549335
4602907
5067319
4739853.667
284855.4865
1
0.5
C-3
3916289
3598140
2612292
3375573.667
679892.5456
0.5
0.25
C-4
3010475
3300069
3147165
3152569.667
144872.6304
0.25
0.125
C-5
2679559
2140249
2722682
2514163.333
324536.3536
0
0
C-6
1958892
1956585
1961565
1959014
2492.240558
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control
Negative Control
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP-BACKGROUND)
PCR Product Concentration (µg/mL)
Total Dilution
Initial PCR Product Concentration (µg/mL)
G6+
3604229
1.604
12
19.248
G6-
2289819
0.29
12
3.48
G6 1-1
1663413
-0.337
12
-4.044
G6 1-2
2506477
0.506
12
6.072
G6 1-3
2619408
0.619
12
7.428
G6 2-1
3001456
1.001
12
12.012
G6 2-2
2163073
0.163
12
1.956
G6 2-3
2329024
0.329
12
3.948
PCR Results: Summary
Our positive control PCR result was 19.248 μg/mL
Our negative control PCR result was 3.48 μg/mL
Observed results
Patient 40032 : All of the pictures taken of this patient's DNA showed very little concentration because the green dye was hardly fluorescent. This is proven by the average concentration of about 6 μg/mL.
Patient 98170 : All of the pictures taken of this patient's DNA showed very little concentration because the green dye was hardly fluorescent. This is proven by the average concentration of about 4 μg/mL.
Conclusions
Patient 40032 : This patient is very similar to the negative control. This patient does not have the SNP associated with the disease.
Patient 98170 : This patient is very similar to the negative control. This patient does not have the SNP associated with the disease.
BME 100 Fall 2017
