Before the experiment the group went over the pre lab reading and the video on the functions of the micropipettor. The video was especially helpful when it came to determining where the first and second stop on the micropipette were, knowing how to prevent bubbles and setting the correct amount of microliters. One member of the group also had previous micropipetting experience which helped immensely when it came down to using the micropipette. The proper materials were gathered before the experiment which included: the patient DNA samples, the micropipette, the PCR reaction mix, and the empty PCR tubes. The empty tubes were labeled positive control, negative control, 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3. The experiment started with putting 50 microliters of PCR reaction mix into the empty positive control tube. Then 50 microliters of the positive control were added to the same tube. This lead to 100 microliters of total solution. These steps were repeated with the negative control, with patient 1's DNA and patient 2's DNA. Once all the solutions were put together correctly, the 8 solutions were placed into the PCR machine. The final reactions ended up all containing 100 microliters of each solution.
Fluorimeter Procedure
Imaging set-up
To properly calibrate the iPhone that was used to capture the images of the solutions, a 160 microliter drop of water was used as a point of reference. This drop was placed on the rough side of the slide which was then inserted onto the fluorimeter and the iPhone was placed on a stand. The fluorimeter was elevated by three plastic trays so that it was held level with the camera. Once this was all set up, it was determined that the correct distance between the iPhone and the fluorimeter for a focused photo was 8cm.
Placing Samples onto the Fluorimeter
Place a slide with the rough side facing up onto the fluorimeter
Set the micropipettor to 80 microliters
Place 80 microliters of SYBR Green I on the rough side of the slide between the first two dots
Place 80 microliters of the solution being tested onto the drop of SYBR Green I
Align the drop with the light on the fluorimeter
Set the iPhone timer to 3 seconds and ensure the iPhone is 8cm from the fluorimeter
Place the black box over both the iPhone and fluorimeter
Activate the camera and lower the flap of the box
Remove the black box and repeat these steps for all trials
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
zero DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL)
FInal DNA concentration in SYBR Green I soulution (micrograms/mL)
Sample Number
RAWINTDEN DROP-BACKGROUND
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
6085564
6082136
8047235
6738311.667
1133562.154
2
1
C-2
8765682
8802422
8806317
8791473.667
22420.9792
1
0.5
C-3
6917230
6678056
6844183
6813156.333
122568.516
0.5
0.25
C-4
5030195
5032233
4993516
5018648
21788.79136
0.25
0.125
C-5
3840540
3841786
3918599
3866975
44712.03597
0
0
C-6
1381272
1423272
1381272
1395272
24248.71131
Calibration curves
(The Calibration Curves do have the error bars placed on the data points, but most of them are too small to be visible on the scale of this graph.) Images of Our PCR Negative and Positive Controls
Negative control PCR
Positive control PCR
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Produce Concentration (micrograms/mL) (Step 5 calculation)
Patient 83982 : Patient 83982's drops did not visibly glow green when placed on the fluorimeter. The samples looked similar to the negative control in the images. The initial concentrations for the three samples were found to be 1.464 μg/mL, 1.2 μg/mL, and 3.12 μg/mL, respectively.
Patient 29244 : Patient 29244's drops glowed a vibrant green color when placed on the fluorimeter. These samples looked extremely similar to the positive control in the images. The initial concentrations for the three samples were found to be 38.508 μg/mL, 37.332 μg/mL, and 38.1 μg/mL, respectively.
Conclusions
Patient 83982 : Since all three initial concentrations of the samples for Patient 83982 (1.464 μg/mL, 1.2 μg/mL, and 3.12 μg/mL) were found to be below the threshold of the negative control's initial concentration of 4.44 μg/mL, it can be concluded that Patient 83982 is negative for the disease.
Patient 29244 : Since all three initial concentrations of the samples for Patient 29244 (38.508 μg/mL, 37.332 μg/mL, and 38.1 μg/mL) were found to be above the threshold of the positive control's initial concentration of 27.564 μg/mL, it can be concluded that Patient 29244 is positive for the disease.
BME 100 Fall 2017
