BME100 f2016:Group1 W1030AM L5
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LAB 5 WRITE-UP
(pictured from left to right: Gabrielle F. Wipper, Carlye A. Frisch)
(pictured: Gabrielle F. Wipper)
PCR Reaction Report
Overall, we found that this lab was a great learning experience in PCR analysis. The pre-lab reading was very helpful. We found that it prepared us well for the lab, especially the pre-lab quizzes where we focused on each lab day's main concepts. After a few trial and errors, we had a much better understanding over the first and second stop on the pipettor; the first stop collects the solution, whereas the second stop releases the solution. The final reactions did have the same amount of liquid and some liquid was left in the tubes with the DNA samples and PCR reaction mix. We did not have to change our labeling scheme throughout this lab.
First, the fluorimeter was set up. A hydrophobic slide was placed onto the device with its smooth side down. The blue LED light was turned on to lighten the first two rows of the slide. A cradle with a smartphone on it was set to take pictures of the slide. The distance between the camera of the smartphone and the first two rows of the slide was adjusted in a way that the camera was focused at that part of the slide and as close to it as possible. It was also important to higher the fluorimeter (in our experiment, we used plastic trays to do so) so that the camera would take pictures of the drops on the slide sideways.
Placing Samples onto the Fluorimeter
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibration Mean Values
Note: Error bars of the standard deviations have been added to both plots, but are invisibly small.
Images of Our PCR Positive and Negative Controls
PCR Results: PCR concentrations solved
The first test for this patient did not show much green in the drop of solution. The second and third test look very similar to this first one as the drop appears to be clear. These tests look similar to the negative control. In the first test for this patient, the PCR product concentration was -1.158860667 μg/mL while the initial concentration was -13.906328 μg/mL. In the second test the PCR product concentration was -.927717335 μg/mL and the initial concentration was -11.13260802 μg/mL. In the third test, the PCR product concentration was -1.261067667 μg/mL and the initial PCR product concentration was -15.132812 μg/mL. The first test's mean we found to be 682278.666, the second's was 1144565.33, and the last one's was 477864.666.
The first, second, and third test appear to be clear solution from the pictures taken. They all look similar in comparison to the negative control solution. In the first test, the PCR product concentration was -.7961925 μg/mL while its initial PRC product concentration was -9.55431 μg/mL. The second test's PCR product concentration was -1.0372295 μg/mL and its initial PCR product concentration was -12.446754 μg/mL. In the third test the PCR product concentration was -1.035125834 μg/mL while the initial PCR product concentration was -12.42151 μg/mL. The first's mean was 1407615, the second's was 925541, and the last one's was 929748.333.
The three test results of the above patient are really similar, the fact which shows the reliability of the test done. The three values are between -11.13 and -15.14, resembling the initial PCR concentration of the negative control (-15.16). However, neither value exceeds the latter value, thus it can't be stated that the patient is truly negative. The test results are nothing like the positive control. The final conclusion made for patient 49774 was negative (as of 11/1/2016). Nevertheless, having further analyzed our data since then, inconclusive would be the accurate judgment.
The relatively small deviation of the results of the three tests demonstrates that the test done was reliable. The values examined are very similar to the results of the samples from patient 49774. In this case neither can it be defined if patient 38123 is truly negative for the test even though the tendency of initial PCR concentrations is surely negative and not positive. Although the final conclusion made previously was negative, the outcome is rather inconclusive in case the concentration of the negative control is considered a valid threshold value.