Within our team, Kami did the majority of the pipetting as she has had prior experience with micropipetting and the process would be more efficient that way. Because she already has a lot of experience with micropipetting, the pre-lab reading didn’t help her much but was very informative to the other group members. The first stop on the pipette pushes out the air is in the tip to create a vacuum that will then suck up the liquid when the button is released, it also sets the load volume. The second stop pushes out all of the liquid in the tip to deposit it, as one passes the first stop, it pushes out the complete amount of liquid in the micropipette, not just the amount the pipette was set to. This helps ensure that the correct amount is expelled if there is any air in the pipette tip. Kami already understood the difference between the first and second stops but the videos helped everyone else in the group understand it as well.
Due to the use of a micropipette, all the final reactions had exactly 100 microliters in the tubes. There wasn’t any liquid left in the tubes that the DNA samples and PCR reaction mix as the starting amount in each tube was approximately 50 microliters so any liquid left over was very minimal and thus not easily visible.
Our labeling scheme remained consistent throughout our investigation and never had to be changed. Michelle’s labels were very neat which helped us not need to change our labeling scheme and stay organized.
Fluorimeter Procedure
Imaging set-up
The set up for capturing the images was as follows. The phone used to capture the images was set on a stand, with the fluorimeter directly in front of it. The height of the fluorimeter was then adjusted by stacking objects under it to ensure the the images captured would be a direct side view of the droplets. In between the fluorimeter and phone stand was a water bottle used to keep the phone stand from falling over when pressed and to keep the distance between the phone and fluorimeter constant. The distance between the camera and the drop was then about 6.7cm. After a drop of a sample was on the slide on the fluorimeter the light switch was turned on and the entire system was covered by the lightbox. The phone camera was set on a ten second timer, and once the image was ready to be captured the camera was activated and the cover of the lightbox was closed while the image was being captured.
Placing Samples onto the Fluorimeter
Before any samples could be placed on the fluorimeter, the slide had to be placed in the appropriate spot on the fluorimeter with the smooth side down and the rough side facing up.
An 80 µL drop of SYBR Green I solution was placed on the slide on the first two clear circles right next to the bulb on the fluorimeter.
An 80 µL drop of the sample or calibration solution was then placed on top of the drop of SYBR Green I.
The slide was then adjusted to ensure that the drop was directly in front of the where the blue beam of light shines.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
1) 5 μg/mL sample
2) 0.5 μg/mL sample
3) zero DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Our positive control PCR result was 19.954904 μg/mL
Our negative control PCR result was 16.34949867 μg/mL
Observed results
Patient 69578: The droplet didn't obviously fluoresce. The average concentration over the three trials was 10.85164 μg/mL.
Patient 58152: The droplet didn't obviously fluoresce. The average concentration over the three trials was 8.932548 μg/mL.
Conclusions
Patient 69578: Negative because the average concentration over the three trials for patient 69578 was closer to the negative control value. In addition, the droplet did not fluoresce in any of the trials.
Patient 58152: Negative because the average concentration over the three trials for patient 58152 was closer to the negative control value. In addition, the droplet did not fluoresce in any of the trials.
BME 100 Fall 2018
