Our team had three members practice pipetting the samples, Connor, Natalie, and Quinten. This was our first time pipetting. The pre-lab reading prepared us sufficiently, by showing us the steps for pipetting. The first stop allowed the DNA mix to be taken into the pipette, while the second stop allowed the mix to be released. The final reactions all had the same amount of liquid, 100 microliters each. There was little to no liquid left in the tubes after the DNA samples and PCR reaction mix were combined. Our labeling scheme stayed the same as what was written before.
Fluorimeter Procedure
Imaging set-up
Set up light box upside down around fluorimeter. The open flap should face the front.
Stack books in order to lift the fluorimeter and DNA sample slides to be level with the smartphone camera.
Using switch, turn on the excitation light for the Blue LED.
Place smartphone at an 90 degree angle from the slide. Adjust the height in order for the camera to take a picture of the drop sideways.
Adjust the distance to at least 4 centimeters between the smartphone and the first two rows of the slide.
Using ruler, record distance between smartphone cradle and drop. Be careful to not move camera.
Placing Samples onto the Fluorimeter
Using the pipettor, place an 80 microliter drop of the SYBR GREEN in the middle of the first two rows of the slides. Then add 80 microliters of one of the calf thymus solutions.
Moving the slide, align the drop so that the Blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
Use camera's timer so that a picture can be taken after covering the fluorimeter and camera with the light box.
With the drop in focus, take three images of the drop.
Remove the 160 microliter drop from the surface using a pipettor, and then move lside to the next position.
Repeat steps 6-11 for the other concentrations of calf thymus DNA.
If a mistake is made, you can use several slides if you make a mistake or want to rerun the calibration.
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 46.030 μg/mL
Our negative control PCR result was 33.840 μg/mL
Observed results
Patient 26963: This patient's PCR values were closer to 40 rather than 30. The calculated numbers ranged from 22.528μg/mL to 115.739μg/mL, skewing on the higher side. Also, the color of the image came out to be a bright green.
Patient 29896: When calculating this patient's PCR, it came out to be a number that is closer to 40 than 30.The numbers calculated similarly ranged from 21.712μg/mL to 100.439 μg/mL. Additionally, the color of the image came out to be a bright green.
Conclusions
Patient 26963: The average of Patient 26963's calculated values was 75.592μg/mL, which is closer to the positive control value of 46.030μg/mL, compared to 33.840μg/mL. Therefore, it can be concluded that this patient is positive. The color of the solution also indicated that the patient was positive by glowing green.
Patient 29896: The average of Patient 29896's calculated values was 66.807μg/mL, which is, again, closer to the positive control value of 46.030μg/mL, compared to 33.840μg/mL. Therefore, this patient is also positive. The solution for this patient also glowed green, indicating a positive result.
BME 100 Fall 2018
