Pipetting was, at first a bit confusing understanding what liquids went in which test tubes. Watching the videos on proper pipetting techniques made the process a bit easier. The pre-lab instructions and quiz helped to understand the main principles of this lab. At first the group wasn't completely sure of what to do, however the videos made everything a lot simpler to understand. The first and second stop of the pipette were easy to understand; the first is for inserting solution into device and the second was to eject what was left off of the device.
The final reactions had the same amount of liquid. The labels stayed the same in order to keep track of each one more efficiently.
Fluorimeter Procedure
Placing samples on fluorimeter
Obtain a tray of sample tubes from the instructors.
Place an 80 microliter drop of SYBR GREEN 1 in the middle of the first two rows of the slide using a micropipette.
Place 80 uL of the sample/calibration solution on top of the SYBR GREEN 1 drop
Align the drop by moving the provided slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting
Place smartphone by the fluorimeter to view the drop in focus about 4 cm away.
Cover smartphone with lightbox, but keep one flap open
Make sure drop is focused again before pressing timer on smartphone
Press timer button on the camera and lower flap before taking the focused picture
Remove the 160 uL drop from the slide and discard liquid waste in hazardous waste container
Move the slide to the next position (center of next two circles)
Repeat above procedures until all five possible measurement positions on the slide have been used.
Imaging set-up
In order to capture a picture of each drop, the samsung device had to be set to the correct exposure, saturation and contrast. For each drop, the extraction light was turned on and placed in the correct position. Once that was done, we turned on the settings on the device to take three consecutive photos of the drop. The device camera was then aligned parallel to the drop in order to capture the image. The device was roughly 3cm away from the drop that way the image was not too blurry. The lightbox was then placed over the set up and the images were captured of each drop.
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Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 28.6502274 μg/mL
Our negative control PCR result was 9.16137466 μg/mL
Observed results
Patient 44954: For patient one, we used 80 μg of both PCR reaction mix and SYBR green, for a total of the drop was 160 μg. For this patient, the G2 1-1 sample appeared illuminated in the image and had a light blue color with a dark blue center forming. The G2 1-2 sample appeared fully illuminated with no dark blue color forming in the drop. The G2 1-3 sample image appeared similar to the G2 1-2 sample being completely illuminated with no dark blue forming.
Patient 75503: For the second patient, we used 80 μg of both PCR reaction mix and SYBR green, for a drop total of 160 μg. This patient, the G2 2-1 appeared fully illuminated with a slight darker colored blue base forming. The G2 2-2 drop was less illuminated with a slight bigger dark blue base forming.
The G2 2-3 sample appeared more illuminated however still maintained a dark blue center taking up a majority of the sample image.
Conclusions
Patient 44954: This patient displayed similar qualities to that of the positive control because of the similarity in the illumination patterns. It maintained a light blue color throughout the experiment. Therefore, this patient is not similar to the negative control because it maintained illumination rather than clouding into a darker non-illuminated color. This patient was successful with the PCR reaction and produced a very illuminated drop.
Patient 75503: This patient was closer to the qualities of the negative control because of the lack of illumination that formed in the drops compared to the patterns of the positive control. This patient is not similar to the positive due to the lack of illumination in the drops. This patient did not have a successful reaction to the PCR as the other patient because the drop was not completely illuminated, therefore the DNA count was less.
BME 100 Spring 2018
