First, the fluorimeter was turned on, and using gloves, the slide was placed into the fluorimeter smooth side down. Then, the cell phone was placed in the cradle, and the fluorimeter was set up on top of tip containers so that the cell phone that was used could reach the top of the fluorimeter to get a camera view of the slide's edge. 80 uL of SYBR Green I and 80uL of the calibration solution were then placed in between the first two dots of the slide, and the light was adjusted to illuminate the center of the drop. The camera was then adjusted to be about 4 centimeters away from the fluorimeter. Then, the black cover was placed around the fluorimeter to make sure that the fluorimeter was protected from light, and the camera was adjusted one last time to make sure that the drop was focused. A 3 second timer was then set up on the camera to take 3 images once set off, the timer was depressed, then the flap on the cover was lowered before the picture was taken. The cover was then taken off, and the the 160 uL of the liquid was discarded into the waste container. The slide was then adjusted to the net row of dots, for the next samples to be placed onto the slide. Repeat above for all calibration solutions.
Placing Samples onto the Fluorimeter
Label the tubes with the buffer (the tubes are marked with red dots) with the positive control, negative control, 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3, which correspond to the patient and trial number.
Use clean pipette tips to place 100 µL of each DNA sample into each corresponding labeled tube with the buffer.
Place 160 uL of water in the center of the first two rows of the slide.
Turn on the light switch for the fluorimeter
Adjust the camera in the cradle to be at a right angle with the side, set off the timer, and close the flap while the camera takes pictures.
Take off the cover, remove the drop of water from the slide, and put the slide smooth side down back in the fluorimeter
Aliquot 80 uL of SYBR Green I to the next row of dots on the slide with a new tip
Aliquot 80 uL of the DNA sample to the SYBR Green I dot, and make sure the light is centered on the dot
Place the cover back around the fluorimeter, adjust the camera, set off the timer, and close the flap
Take the cover off, remove the 160 uL on the slide
Repeat above steps for the rest of the DNA samples
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA
Low Calf Thymus DNA
Zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control PCR Sample
Positive Control PCR Sample
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 131.52 μg/mL
Our negative control PCR result was 112.57 μg/mL
Observed results
Patient 45783: The droplets all had a very bright, fluorescent green light shining through the droplet, and the average initial PCR product concentration was 97.38 μg/mL.
Patient 21721: The droplets were all clear and had little to no light shining through them, and the average initial PCR product concentration was 16.28 μg/mL.
Conclusions
Patient 45783: Since the positive droplet had a very bright green color similar to this patient’s droplets, and the average initial PCR product concentration was 97.38 μg/mL which is a higher number, we came to the conclusion that the patient’s DNA tested positive.
Patient 21721: Since the negative droplet was very clear which was similar to this patient’s droplets, and the average initial PCR product concentration was 16.28 μg/mL which is a lower number, we came to the conclusion that the patient’s DNA tested negative.
BME 100 Spring 2017
