Our team was successfully able to carry out the PCR reaction. We had previous experience working with micro-pipettes, so we were good at differentiating between the first stop and the second stop. The first stop was used to suck up liquid, and the second stop was used to dispel it. The final reactions all had 100 micro-liters of liquid, and all the tubes looked like they contained the same size sample. They should have all had the same amount of liquid, because we were careful not to get bubbles when pipetting the samples. The pre-lab reading helped to clarify the order of the steps, meaning whether or not the reaction mix or the sample should be added first. There did not appear to be liquid left in the sample tubes, but there may have been some small drops due to human error. Our labeling scheme was successful because our labels were only a few characters long, and the tubes did not have much room to write more on them.
Fluorimeter Procedure
Imaging set-up
First the glass slide was placed into the fluorimeter with the smooth side down, and it was placed onto a plastic box to raise it up higher. Then the blue LED light was turned on, and 160 micro-liters of water was placed onto the glass slide in between the first and second row of the light. The smartphone cradle was placed in front of the fluorimeter, and the smartphone was placed so that it viewed the water droplet from the side, parallel to the LED light. The distance between the smartphone and the drop was adjusted until the smartphone could properly adjust to the drop, and this distance was recorded. The camera was adjusted, and a timer was set for 10 seconds. After starting the camera timer, the box was placed over the fluorimeter smartphone system to keep as much light as possible out of the picture. To set up the camera, turn flash off, set ISO to 800 if possible, increase exposure and saturation to the highest level, set white balance to auto, and decrease the contrast.
Placing Samples onto the Fluorimeter
With gloves, place glass slide, smooth side down, on fluorimeter, and turn it on
Place smartphone in cradle, and place it to the edge of the fluorimeter, parallel to the LED light
Adjust height of fluorimeter until the camera views the glass slide from the side, instead of from above
Focus camera, change camera settings, and measure distance between LED light and smartphone camera
Place disposable top on micro-pipettor
Push to first stop and suction 80 micro-liters of SYBR green 1 solution by letting go of first stop, and place onto middle row between LED light by pushing to second stop
Dispose tip, replace with a new tip
Push to first stop and suction 80 micro-liters of one of the calf thymus solutions and place on top of SYBR green 1 drop by pushing to second stop
Focus camera on drop and place box over the fluorimeter, with one flap open
Start timer, and close flap while pictures are being taken
Remove box and suction off of the slide after taking the pictures by pressing to first stop and releasing
Dispose of liquid in disposable waste cup
Repeat steps 1-6 with the rest of the calf thymus solutions, but change which part of the glass slide the drop is placed onto by putting a new slide onto the fluorimeter
Now all the steps should be repeated, but instead of using the calf thymus solutions, 80 micro-liters of each of the PCR reaction samples should be used
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Zero Calf Thymus DNA (0 μg/mL sample):
Low Calf Thymus DNA (0.5 μg/mL sample):
High Calf Thymus DNA (5 μg/mL sample):
Calibrator Mean Values
Sample Number
Image number
Final DNA concentration in SYBR Green I solution (µg/mL)
To the eye, the images for this patient looked brighter and closer to the positive control than the images for the other patient. These images have a greener tint.
Patient 76146: -55.60 μg/mL
In comparison, these images are more dim and blue tinted.
Conclusions
Patient 29486: Positive
Patient 76146: Negative
For both patients, the data was too inconsistent to provide a definitive positive or negative reading. All of the concentrations came back with negative values, which is theoretically impossible. Errors were made either with the PCR reaction, the fluorimeter, or the imageJ analysis. However, to the naked eye, the first patient's images were brighter and more similar to the positive control than the second patient's, leading to the conclusion that the first patient is positive and the second patient is negative. However, in order to confirm these conclusions further testing must be done.
BME 100 Spring 2018
