Overall, the team’s experience with pipetting the samples to set up the reaction was positive. The pre-lab reading helped because it made it easier to understand what was going on. For example, the difference between the first and second stop on the pipettor. The first stop of the pipettor is used to collect the sample and the second stop is for releasing the collected sample.
It was found that the final reactions had exactly the same amount of liquid and 20.0 µL of liquid was left in the tubes that the DNA samples and PCR reaction mixed in. Overall, the labeling scheme was not changed during the experiment.
Pictured: Angie Quinonez (Left), Lesly Romero (Center), Gisselle Beltran (Right) next to the PCR machine. (Photo Credit: Jenny Wong)Pictured: Mason Haynie (Left) and John Nguyen (Right) micropipetting. (Photo Credit: Jenny Wong)
Fluorimeter Procedure
Imaging Set-Up
In order to take images of the experiment being conducted there were different components and steps that had to be put together. The model of the smart phone used was and iPhone X. The phone's camera settings were set to match that of the procedures below. By placing the phone vertically on the cradle we were able to capture our necessary pictures.
Placing Samples onto the Fluorimeter
Place a 160 microliter drop of water in the middle of the first two rows of the slide with the micropipettor. The drop should be pinned and look like a beach ball.
Using the switch for the Blue LED turn on the excitation light.
Turn on the camera of your smartphone. If you can change the settings on your smartphone change the settings to be... -Set the flash to inactive -Set ISO to 800 (or higher, if possible) -Set white balance to auto -Set exposure to highest setting -Set saturation to the highest setting -Set contrast to lowest setting
Place your smartphone on the cradle at the right angle for the slide.
Adjust the height of the fluorimeter using the plastic trays so that your camera takes a picture of the drop sideways.
Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as possible without making the image blurry. -Note: It should be at least 4 centimeters away from the drop.
Record the distance between your smartphone cradle and drop using the ruler provided in lab. -Note: Be careful not to move the camera, cradle, or fluorimeter because the light collected will change slightly if there is a significant difference from one image to the next in these distances.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Sample: 5 μg/mL
Sample: 5 μg/mL
Sample: 0.5 μg/mL
Sample: 0.5 μg/mL
Sample: Zero DNA
Sample: Zero DNA
Sample: 5 μg/mL (with circle)
Sample: 5 μg/mL (with circle)
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL)
Final DNA Concentration in SYBR Green I Solution (μg/mL)
Our positive control PCR result was 195.3911854 μg/mL.
Our negative control PCR result was 46.27021388 μg/mL.
Observed Results:
Patient 83872: After making some observations, the patient had a sample that looked a lot more similar to that of the negative than the positive. The patient's images showed some green colors present. This signified that there was the positive sample present because those are the same qualities represented. In regards to the quantitative data, the Initial PCR Product Concentration (µg/mL) were closer to the value of the negative control. The data of 41.60823262 µg/mL, 40.69406982 µg/mL, and 36.1034669 µg/mL appeared to be closer to the negative control value of 46.27021388 μg/mL than the positive control value of 195.3911854 μg/mL. Therefore, it was determined that the patient was negative.
Patient 97753: After making observations for Patient 2 we noted that these patient's sample looked more similar to that of the negative control. There was not much green present and in order to show positive then there would have had to have been more green. There was a dot that was present in the middle of the sample but there was not green present. For the quantitative data, we had an outlier that was much greater than the other numbers therefore we predict that this could have an effect on our data. This could possibly be due to the fact that we are new users to this technology and this could have led to human error. In regards to the quantitative data, the Initial PCR Product Concentration (µg/mL) were closer to the value of the negative control. The data of 34.47776937 µg/mL, 51.91755691 µg/mL, and 203.9632886 µg/mL, appeared to be closer to the negative control value of 46.27021388 μg/mL than the positive control value of 195.3911854 μg/mL (when disregarding the outlier of 203.9632886 from trial 2-3). Therefore, it was determined that the patient was negative.
Conclusions:
Patient 83872: The data of 41.60823262 µg/mL, 40.69406982 µg/mL, and 36.1034669 µg/mL appeared to be closer to the negative control value of 46.27021388 μg/mL than the positive control value of 195.3911854 μg/mL. Therefore, it was determined that the patient was negative. The qualitative data also demonstrated these factors because the lack of green showed negative.
Patient 97753: The data of 34.47776937 µg/mL, 51.91755691 µg/mL, and 203.9632886 µg/mL, appeared to be closer to the negative control value of 46.27021388 μg/mL than the positive control value of 195.3911854 μg/mL (when disregarding the outlier of 203.9632886 from trial 2-3). Due to the fact that the average of person 2 was closer to that of the negative we concluded that this patient is negative. The qualitative data also demonstrated these same results because there was no green in the images which is a sign showing negative.
BME 100 Fall 2018
