Initially using the micropipettes presented some small challenges. As explained in the pre-lab informational video, it is important to recognize the difference between the first and second stops when dispensing solution. While we were aware of this difference and used caution while handling the equipment, the most effective way to learn the proper use of micropipettes was to physically practice with them. While learning the proper way to operate the pipettes, some group members struggled with dispensing the full amount of solution -- not always pushing the dispense button to the second stop and sometimes releasing while the micropipette tip was still in the solution, causing air bubbles and retraction of small amounts of solution. After overcoming these learning curves, the group was able to work out an effective method of using the micropipette technology.
Fluorimeter Procedure
Imaging set-up
We set up the box that contained the device in order to cover it completely, leaving one side open to place our camera stand in. We then set a three second timer on our camera so once we pressed the button to take the picture we had time to close the box, making a completely dark space, and optimizing the image captured of the fluorimeter.
Placing Samples onto the Fluorimeter
Remove the flourimeter from the shadow box.
Slide the glass into the two grooves of the flourimeter with the smooth side facing up.
To begin attach a clean tip to the pipette.
Then pipette exactly 80 mL of the SYBR Green and dispense it on the fluorimeter. The liquid should be placed so the light can reflect off of the droplet.
Next dispense the tip into the beaker and attach a new clean tip.
Pipette exactly 80 mL of one of the 8 solutions that were prepared in Lab C. This liquid should be added on top of the droplet that was already on the fluorimeter.
Then dispose of the tip into the beaker. A new clean tip should be added to the pipette.
Set the phone's camera to take a picture 3 seconds after being pressed and position it on the stand so the droplet is the main focus and can be seen clearly with the light. Also ensure that the phone's flash is turned off before being positioned on the phone stand.
The shadow box should be put over the flourimeter and phone set up, and take three pictures of the droplet in complete darkness.
Carefully remove the shadow box and suck up the droplet off of the flourimeter using a pipette. The tip is then disposed of.
Repeat steps 3 through 10 for the next seven solutions.
When finished with all 8 solutions, properly dispose of all of the tips and flourimeters in the red bins and trash cans.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
0 μg/mL sample
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Patient 96033 : The first patient's images looked pretty dim in all three sets, and the initial PCR values have a lower mean than those of patient 2.
Patient 38209 : The second patient's images looked much brighter in all three sets, and the initial PCR values appear to have a higher mean.
Conclusions
Patient 96033 : We believe our first patient tested negative for this disease due to the lack of fluorescence observed as well as our quantitative observations.
Patient 38209 : We believe our second patient tested positive for this disease due to the greater amount of fluorescence we observed as well as our quantitative observations.
BME 100 Fall 2018
.jpg)
