The overall experience with the pipetting was quite simple. The pre-lab material was helpful in order to properly determine how to set the micropipette to 50 microliters, how to attach the pipette tips, and how extract solutions from the test tubes. We understood the difference between the first stop and the second. The first stop is used to properly fill the pipette tip with the 50 microliters whereas the second stop is useful to expel all the solution from the pipette tip into a new test tube. The final reactions had approximately the same amount of liquid with the help of the first stop of the micropipette. There were a few air bubbles and some liquid left in the pipette tips after expelling the liquid. We did not have to change out labeling scheme. We kept true to the chart with the correct group numbers and DNA samples as seen in our previous lab. The first sample of DNA was used for G2 1:1-3 and the second sample of DNA was used for G2 2:1-3.
Fluorimeter Procedure
Imaging set-up
In order to use the non-standard, drop fluorimeter, our lab group turned on the device. We placed the glass slide on the fluorimeter, smooth side down. Then we adjusted the height of the device so that the camera view is directly pointed at the slide. We placed the iPhone in the cradle, and we placed it 5 centimeters away from the base of the fluorimeter. We, then, placed the black-out light box on top of the system. Next, we lifted the light box door to press the capture button on the camera and then we closed the door so that the camera could capture the picture in complete darkness.
Placing Samples onto the Fluorimeter
Place a 160 microliter drop of water in the middle of the first two rows of the slide using the pipettor.
Turn on the excitation light using the switch for the Blue LED.
Turn on the camera of your smart phone and check the settings on the smartphone
Place the smartphone on the cradle and adjust the height of the fluorimeter so that the camera takes a sideways picture.
Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is 5 centimeters away.
Place a 80 microliter drop of SYBR GREEN I in the middle of the first two rows of the slide using the pipettor, then add 80 microliters of one of the calf thymus (or water blank) solutions
Focus the light so that it aligns with the drop of solution.
Use the timer on the camera to take a picture while the fluorimeter and camera are covered with the light box.
Take three images of the drop.
Remove the box and readjust the position of the phone and cradle.
Use the pipettor to remove the 160 microliter drop from the surface and move the slide to the next position.
Repeat steps 6 - 11 for the other concentrations of calf thymus DNA.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
1. 5 μg/mL sample
2. 0.5 μg/mL sample
3. zero μg/mL sample
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA Solution (Micrograms/mL)
Final DNA Concentration in SBYR Green I Solution (Micrograms/mL)
Sample Number
Image 1
Image 2
Image 3
Mean
Standard Deviation
5.00
2.500
C-1
15,575,017
15,242,389
15,979,506
15,598,971
369,142
2.00
1.000
C-2
11,508,609
11,289,890
10,612,219
11,136,906
467,367
1.00
0.500
C-3
10,424,507
10,424,507
9,860,749
10,111,538
286,977
0.50
0.250
C-4
9,142,734
9,142,734
8,545,604
8,554,050
584,507
0.25
0.125
C-5
7,795,886
7,795,886
7,241,671
7,449,043
302,287
0.00
0.000
C-6
6,437,471
6,116,958
5,794,971
6,116,467
321,250
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 (Micrograms/mL)
Total Dilution
Initial PCR Production
Concentration
(Micrograms/mL)
G2 1-1
13,167,678
2,194,612
12
26,335,344
G2 1-2
12,110,572
2,018,428
12
24,221,136
G2 1-3
15,122,480
2,520,412
12
30,244,944
G2 2-1
3,692,539
615,422
12
7,385,064
G2 2-2
2,988,471
498,078
12
5,976,936
G2 2-3
3,290,174
548,361
12
6,580,332
(-) Control
3,311,241
551,873
12
6,622,476
(+) Control
13,454,783
2,242,063
12
26,909,556
PCR Results: Summary
Our positive control PCR result was 26909556 μg/mL
Our negative control PCR result was 6622476 μg/mL
Observed results
Patient 1-ID: 95353 : The images for the first patient showed green florescent drops. The green color had the same visual intensity as the positive control sample our group imaged. The average of the microgram/mL of the first patient is 26,933,808.
Patient 2-ID: 64816 : The images for the second patient showed drops with no significant florescent color. The drops had a slight purple hue, but it was not bright or intense. The images for our second patient modeled the same color and lack of intensity and florescence as the negative control provided. The average of the three samples of the second patient is 6,647,444 micrograms/mL
Conclusions
Patient 95353 : Positive Conclusion
Our lab group concluded with the positive result because of its qualitative and quantitative similarities to the positive control
Patient 64816 : Negative Conclusion
Our lab group concluded with a negative results because of its qualitative and quantitative similarities to the negative control
BME 100 Fall 2016
