The pre-lab reading was very helpful. The videos show step by step how to use a pipette and tips for having successful transfers of liquids. When using a pipette, it is important to understand the difference between the first and second stop. The first stop on the pipette is used for retaining liquid. Once the desired amount has been set, place the pipette into the liquid and push down slowly to the first stop. This will allow the exact amount to be contained. The second stop is used to dispense the liquid and should be pressed down slowly as well. The final reactions lost roughly 5 uL from it's original 100uL. The extraction of both the DNA samples and the PCR reactions left no extra liquid in the tubes. All of the samples were used in the experiment. The labeling scheme for the tubes stayed the same throughout.
Fluorimeter Procedure
Imaging set-up
In order to properly set up the camera, the cradle was positioned 4 centimeters away from the flourimeter. To get the most accurate results, the camera lens should be at eye level to get the best look at the SYBR Green and the drop. The drop should be propped up on small boxes to be at eye level with the camera lens to ensure good quality photos. Finally, in order to eliminate all white light from the fluorimeter, a folder was used to block the light on the open side of the box to ensure the best SYBR Green data.
Distance between the iPhone camera and drop = 4.0 cm
Smart Phone Settings
Type of Phone: iPhone 6+
Flash: Off
White Balance: Auto
Exposure: Highest
Saturation: Highest
Contrast: Lowest
Placing Samples onto the Fluorimeter
A tray was obtained containing 8 tubes of buffer, 2 tubes of SYBR Green 1, 1 tube of pH 8 water, and 5 tubes containining Calf Thymus DNA.
A 160 μL drop of water was pipetted into the center of the first two rows of the slide, the smartphone camera was set up at a right angle to the slide, and three images of the water drop were taken. The distance between the cell phone and the droplet was recorded for future trials.
80μL of SYBR Green 1 was placed in the middle of the first two rows of the slide, and 80μL of the first DNA sample was added. Three images of this drop was taken.
The micropipettor was used to remove the 160μL droplet, and step 3 was repeated on the next slide position using the next DNA sample.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
1. 0 μg/mL
2. 0.5 μg/mL
3. 5 μg/mL
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 69.55 μg/mL
Our negative control PCR result was 66.33 μg/mL
Observed results
Patient 22171 : The images for patient 1 showed some green under the light and were white(light) in each drop in Image J. Quantitatively, the results showed that on average the initial concentration was 63.91 μg/mL.
Patient 64572 : Qualitatively, patient two's images showed a nicely sized green dot in the center of each drop. Quantitatively, the results showed that on average the initial concentration was 63.32 μg/mL.
Conclusions
Patient 22171 : All of the samples for patient one were closer to the negative control values than they were for the positive control, so it can be concluded that patient one is negative.
Patient 64572 : While two of the samples for patient two were closer to the negative control, one sample almost exactly matched the positive control value, and these patient's samples were similar to the positive control when performing gel electrophoresis. The PCR results are inconclusive, but the gel electrophoresis results led to a conclusion that this patient is positive.
BME 100 Spring 2017
