Pre-lab reading allowed us to be better prepared in micropipetting. First and second stop on the pipettor were clearly distinguishable. We did not need to change our labeling scheme.
Prepared samples for patient 45247 and patient 78801 with positive and negative controls. Each tube with positive, negative, 45247 or 78801 was mixed with DNA/Primer mix. Each type of sample was contained 50 microliter of control or patient replicate DNA combined with DNA/Primer mix resulting in 100microliters of total fluid.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy 5S
Flash: Off
ISO setting: 800
White Balance: Auto
Exposure: +2.0 (highest setting)
Saturation: n/a
Contrast: n/a
Camera set-up
We place the smartphone inside the camera cradle 9 cm away from the droplet with the camera settings as mentioned above. Because the smartphone camera was a lot taller than the fluorimeter, adjustments to increase its height must be done. We placed two plastic boxes underneath the fluorimeter to have it be leveled to the smartphone's camera.
Distance between the smart phone cradle and drop = 9cm
Placing Samples onto the Fluorimeter
Place a slide directly into the fluorimeter slot, smooth side down
Without moving the phone position or the fluorimeter micropipette 80 microliters of SYBR Green solution onto the middle of the first two rows of slide cells.
Similarly, do the same with CALF THYMUS solution: micropipette 80 microliters onto the SYBR Green already on the slide, creating a total of 160 microliters
Move the "light box" into position.
Depress the timer and quickly shut the light box, taking adequate time for the photograph to be taken.
Repeat steps 1-5 with all varying concentrations of CALF THYMUS, keeping SYBR Green as a constant. Take 3 photos of each mixed concentration.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample image (High Calf Thymus)
0.5 μg/mL sample image (Low Calf Thymus)
0 μg/mL sample image (Zero Calf Thymus)
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Negative Controls
PCR Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 18.681 μg/mL
Our negative control PCR result was 1.540 μg/mL
Observed results
PCR Patient 45247 Droplet
PCR Patient 78801 Droplet
Patient 45247: The droplet appeared dark blue, similar to the negative control sample, under the light. The average concentration of the three samples from Patient 45247 was 1.665 μg/mL.
Patient 78801: The droplet appeared dark blue, similar to the negative control sample, under the light. The average concentration of the three samples from Patient 78801 was .520 μg/mL.
Conclusions
Patient 45247: The conclusion was reached that the patient tested negative due to the similarity in the concentrations found in the patient's samples and those found in the negative sample. The imaging also lacked evidence of any green glow under the light that would have been evidence of replication of the targeted sequence.
Patient 78801: This patient also tested negative based on calculated concentration of the DNA and observations from imaging.
BME 100 Fall 2015
