Our team's experience with pipetting the samples was one that was quite fun. It was a first for many using the pipette which gave room for some unexpected errors of dropping sample tubes, however, no samples were wasted. The pre-lab helped very much in minimizing mistakes and helping our team understand what to do and how to do it accurately and efficiently. Discovering the difference between the first and second stop on the pipettor was difficult at first but thankfully, a team member figured the process out and was able to explain it to the rest of the team. There was one sample that seemed to have slightly less liquid compared to the others. The team's conclusion was that lack of experience in pipetting allowed for a team member to accidentally waste liquid that was still left in the pipette tip. This mistake was the only noticeable one that could have affected the results. After careful examination of the tubes that originally contained the DNA samples and PCR reaction mix, there seemed to be no liquid left in any of the tubes. Finally, our team accurately labeled the samples using clear handwriting and obvious labeling to minimize reading mistakes.
Fluorimeter Procedure
Imaging set-up
Step 1: Using water, place a 160 microliter drop in the middle of the first two rows of the slide on the unsmooth side of the slide. The ball should look rounded and almost like a circle.
Step 2: The excitation light is then turned on using the blue LED light.
Step 3: A smartphone is obtained that can adjust exposure and contrast.
Step 4: The smartphone should be placed in the phone cradle and the height of the Fluorimeter can be adjusted using plastic trays to take a picture of the drop from the side.
Step 5: The distance between the smartphone and fluorimeter can be adjusted so that the drop sample of water no longer looks blurry. Make sure the phone cradle is at least 4 centimeters away.
Step 6: Measure the distance between the smartphone cradle and the drop sample using a ruler. Make sure to not move the camera, cradle or fluorometer extensively so as to not adjust the light exposure in the images.
Placing Samples onto the Fluorimeter
Step 1: Using the micropipette, place an 80 microliter drop of SYBER GREEN I on the unsmooth side of the slides. The drop should be in the middle of the first two rows of holes on the slides. Another 80 microliters are then added containing the calf thymus solutions (5, 2, 1, 0.5, 0.25, 0). This drop is a sample.
Step 2: Make sure the slide aligns with the blue LED light on the Fluorimeter. If not, adjust as needed.
Step 3: Make sure the flash is off on the camera and cover the fluorimeter and camera with the black lightbox. The light box is used to remove as much light as possible.
Step 4: Take three images of the drop sample making sure that the drop is focused and not blurry and making sure to label the images as each one is taken.
Step 5: Remove the box carefully making sure to not adjust the camera or the Fluorimeter.
Step 6: The micropipette is then used to remove the 160 microliter drop sample and then repeat steps 1 through 6 for each sample containing the calf thymus solutions (5, 2, 1, 0.5, 0.25, 0) and then the PCR samples (1-1, 1-2, 1-3, 2-1, 2-2, 2-3, +, -).
Step 7: The three images of each sample will then be used to create a dot plot.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA
Low Calf Thymus DNA
Zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control
Negative Control
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 1.97 μg/mL
Our negative control PCR result was -.861 μg/mL
Observed results
Patient 76522 : In the first patient, the green SYBR Green I was not illuminated as much as it could have been, it looked very blue in comparison to the positive control, and this relates to its average PCR product final concentration of -.085, or almost zero. Obviously there was some error involved, due to the line of best fit being skewed by inconsistent values, but it is easy to see that this a number very comparative to zero.
Patient 79360 : In this second patient, the green SYBR Green I was illuminated apparently and was very similar to the look of the positive control. This is supported by the average PCR product final concentration of 2.82, relative to the concentration calculated for the positive control (1.97).
Conclusions
Patient 76522 : negative, the PCR product concentration was very averaged very close to zero and the SYBR Green I was not as apparent as it should have been for a positive result.
Patient 79360: positive, the PCR product concentration was very close to the positive control value, and the SYBR Green I was very apparently illuminated during testing.
BME 100 Fall 2017
