Micropipettes are used to measure and deliver accurate volumes of samples. The pre-lab reading was useful and thoroughly explained the process of using a micropipette, including the calibration as well as the first and second stops. The first stop is used to create a suction for the sample. Then, the second stop is used to dispense of the sample collected. The final reactions did have the same amounts of liquid (80 mL of SYBR Green + 80 mL of the DNA sample). When combining the DNA sample and PCR mix, there was no left liquid. The labeling scheme used did not need to be altered because accuracy was achieved.
Fluorimeter Procedure
Imaging set-up
To set up the fluorimeter for testing, a glass slide was inserted with the smooth side down, and rough hydrophobic side facing up. A smart phone (iPhone 6s) was then set up on a cradle roughly 5 centimeters (cm) away from the fluorimeter. Various camera settings were altered in order to capture the best image. The ISO was set to 800, and the exposure, as well as saturation, was set to the highest setting, while contrast was set to the lowest setting. White balance remained on auto. The fluorimeter was set on top of the box of 69 micropipette tips. A light box was placed around the fluorimeter to cancel exterior lighting. An image was then captured using the smart phone with a 3 second timer under the light box. Flash was inactivated. Although, the smartphone used had an automatic flash during the timer, the image captured was not affected.
Placing Samples onto the Fluorimeter
The micropipette was set to 80 µl.
Using a new micropipette tip, an 80 microliters (µl) drop of SYBR GREEN was placed on the rough side of the slide, in between two circles in a row. The micropipette tip was then discarded.
Using a new micropipette tip, an 80 µl sized drop of sample (calf thymus or DNA) was added.
The fluorimeter was turned on.
The slide was adjusted so the blue LED light from the fluorimeter focused by the middle of the drop.
The light box was then placed over the fluorimeter to cancel out room light.
The smartphone cradle was placed 5 cm away from the fluorimeter.
A timer on the camera of the smartphone was set to 3 seconds (sec).
The capture image button was pressed, and the lid to the light box was shut to achieve complete darkness inside the box.
Two additional images were captured of the drop on the fluorimeter.
The above procedures were repeated for all calf thymus and DNA samples.
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 solution (µg/mL)
Our negative control PCR result was 9.6E(-7) μg/mL
Observed results
Patient 28692: The droplet remained clear; no green was detected once the LED light was focused on the middle of the drop of DNA plus SYBR Green. The concentration of patient 28692 averaged to 7 μg/mL resulting in this patient testing negative.
Patient 68634: The droplet showed green under the PCR reaction; green was detected once the LED light was focused on the middle of the drop of DNA plus SYBR Green. The concentration of patient 68634 averaged to around 30 μg/mL, resulting in this patient testing positive. For the first trial, patient 68634's concentration was closer to the negative control. However, the remaining two trials resulted in concentrations closer to the positive control, resulting in an overall positive result.
Conclusions
Patient 28692: This patient tested negative for the disease single-nucleotide polymorphism (SNP).
Patient 68634: This patient tested positive for the disease single-nucleotide polymorphism (SNP).
BME 100 Fall 2017
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