The pre-lab reading was helpful to understand just how the lab needed to be setup. A few of us have experience with using the micropipettors, so we knew what the first and second stops meant already. There was some liquid left in the tubes of the DNA samples and the PCR reaction mix, and one of the reactions had slightly less liquid in it than the rest. The labeling scheme worked fine and didn't need to be changed.
Fluorimeter Procedure
Web camera set-up
For this lab a phone camera was used instead of a webcam. The phone was placed in the cradle standing nearly vertically. The flourimeter was then placed on some small plates to lift it up to the camera. The height of the flourimeter was adjusted using more or less plates so that the camera could take a picture directly on the side of the drop. The camera was then set to use a timer.
Placing Samples onto the Fluorimeter
With gloves, find the smooth side of the slide and place it on the flourimeter smooth side down.
Set the camera in the cradle and set the timer on it.
Change the height of the flourimeter so the camera can take a picture of the side of the drop of the solution.
Use 80 microliters of SYBR Green I solution and place a drop on the first 2 clear circles in the middle of the slide.
Now place 80 microliters of the sample/calibration solution on top of the SYBR Green I drop.
Move the slide so that the light from the flourimeter illuminates the middle of the drop and focuses on the other side.
Change the distance of the camera so that it takes a close picture of the drop and remains in focus, then record that distance.
Cover the setup with the lightbox and make sure the drop is in focus.
Now press the timer on the camera and close the box with the other flap.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA 0.5 DNA
5DNA
No DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Sample Number
RAWINTDEN DROP - BACKGROUND
'
'
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
17960676
17361666
17432393
17584911.67
327337.2921
2
1
C-2
18302216
18948578
13833067
17027953.67
2785663.578
1
0.5
C-3
16029494
17146613
16272576
16482894.33
587506.6519
0.5
0.25
C-4
10520769
11682263
12226176
11476402.67
871141.2724
0.25
0.125
C-5
6119828
7453912
6946432
6840057.333
673373.3786
0
0
C-6
2816767
2841332
2611419
2756506
126247.9227
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control
Positive 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)
G11 +
3744312.6667
-0.322241048
12
-3.866892571
G11 -
954898.6667
-0.720728762
12
-8.648745143
G11 1-1
1533861.0000
-0.638019857
12
-7.656238286
G11 1-2
1191914.3333
-0.686869381
12
-8.242432571
G11 1-3
1426572.0000
-0.653346857
12
-7.840162286
G11 2-1
1580550.3333
-0.631349952
12
-7.576199429
G11 2-2
1403742.3333
-0.656608238
12
-7.879298857
G11 2-3
1577947.3333
-0.63172181
12
-7.580661714
PCR Results: Summary
Our positive control PCR result was -3.866892571 μg/mL
Our negative control PCR result was -8.648745143 μg/mL
Observed results
Patient 87806 : The image showed a drops that were faintly illuminated. It also had an average of -7.013 μg/mL.
Patient 99993 : The images for his patient were not illuminated and had an average of -7.679 μg/mL.
Conclusions
Patient 87806 : This patient returned a negative results because its PCR results most resembled the negative control.
Patient 99993 : This patient also returned a negative result for the same reason, its PCR results also resembled the negative control.
BME 100 Spring 2016
