Before beginning the lab, our group read the pre-lab information in order to familiarize ourselves with the pipetting techniques and the materials that were used. There was no problem in distinguishing the difference between the first and second stop on the pipettor, the first was used for drawing the liquid and the second was for releasing it.There were eight tubes in total, with 2 controls (positive and negative) and 6 with PCR solutions, and all of these were labeled correctly in the previous lab. By being very precise with our pipetting, the final reactions were able to have the same amount of liquid, and there was no visible liquid left in the tubes. Our labeling scheme was not changed from the previous lab session because there was no need to. Our labeling was clear and easy to understand for our group.
Fluorimeter Procedure
Web camera set-up
Phone camera set up
A smart phone camera was considered a well enough camera to take photos for this lab. There are several changes that should be done before taking pictures; these included, deactivating the flash, changing the ISO to 800 or higher, setting white balance to auto, setting both exposure and saturation to the maximum, and setting contrast to the minimum. In addition, the timer should be adjusted to three seconds. Then, the phone should be placed on the cradle at a right angle. If the angle was not right, adjust the height of the fluorimeter. The distance between the smartphone and the drop was measured and kept at a constant of 9 cm.
Placing Samples onto the Fluorimeter
Place the glass slide with the smooth side facing down on the fluorimeter.
Place the camera phone into the phone cradle and set the timer to three seconds.
Set the micropipette to 80 μL and add 80 μL of the SYBR Green solution to a dot on the glass slide. The solution should be in a ball on the glass slide.
Eject the micropipette tip into the waste cup and replace with a new tip.
Add 80 μL of correct sample to the existing drop of SYBR Green on the slide.
Allow the blue light to shine through the drop of solution on the slide.
Close the flap on the dark-box and set the timer on the phone. Take three photos of the solution.
Remove all of the solution from the slide.
Repeat steps 3-8 for each sample that requires testing.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High:
Low:
Zero:
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive:
Negative:
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 24.119097 μg/mL
Our negative control PCR result was 5.662217 μg/mL
Observed results
Patient 12678 : The images that were obtained for this patient were similar to that of our positive control, as they exhibited a bright green glow. Also, this patient's calculated DNA concentration in all three trials was close to the positive controls's calculated DNA concentration of 24.119 micro liters.
Patient 53171 : The images that were obtained for this patient were similar to that of our negative control, as they exhibited a clear blue glow. Also, this patient's calculated DNA concentration in all three experiments was close to the negative control's calculated DNA concentration of 5.662 micro liter.
Conclusions
Patient 12678 : Because the patient's pictures exhibited a bright green glow indicative of a positive sample and also had DNA concentrations that correlated closely to that of the positive control, this patient is found to be positive for Non- Hodgkin's Lymphoma.
Patient 53171 : Because the patient's pictures exhibited a clear blue glow indicative of a negative sample and also had DNA concentrations that correlated closely to that of the negative control, this patient is found to be negative for Non-Hodgkin's Lymphoma.
BME 100 Spring 2016
