The experience with the pipet with our team was a learning experience. The person who was using the pipet had already used the pipet before and understood the difference between the first and second stop on the pipet. The first stop is to get the majority of the substance out while the second stop provides a little more push, to expel the last drop. With the small amounts that were being used, it was essential to get every drop possible. The pipet tip mechanism was something that was bumped while the liquid was still in the pipet tip, so the tip came off, providing that sample to be void. The pre-lab reading helped because the micropipet is something that is very easy to use, once you know everything that is included and operating as well as it being fragile. The final reactions has the same amount of liquid because the micropipet is a very accurate tool and it was set to the right amount of substance. There was not any liquid left in the reaction mix or the samples, so the provided materials must have been just enough.
Fluorimeter Procedure
Imaging set-up
Place the smartphone onto the phone stand, in order to see how figure out how much the fluorimeter needs to be adjusted higher
Next use an extra test tube stand to prop the fluorometer higher so that the smartphone has a good view from the side
Repeat step two until the fluorometer has reached a desirable height to the smartphone camera
Once the drops have been placed on the fluorometer, take the picture under the box that the fluorometer came in by closing the flap and setting the timer on the smartphone camera (this is because the SYBR Green I solution is sensitive when exposed to light for too long)
Placing Samples onto the Fluorimeter
Place the slide within the fluorometer with the glass side down and the rough hydrophobic side up
Use the micropipette to place 80 µL of the SYBR Green I solution onto the middle two vertical circles on the slide
Use the micropipette to place 80 µL of the diluted PCR solution on to the drop of SYBR Green I solution placed before it
Then flip the switch on the right side of the fluorometer which will illuminate the blue light that must be aligned with the drops of solution
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Left to right: high, low and zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 0.81397 μg/mL
Our negative control PCR result was -1.1449 μg/mL
Observed results
Patient 70416 : The drops for G51-1, G51-2 and G51-3 have green fluorescence showing in them.
Patient 90490 : The drops for G52-1, G52-2 and G52-3 were clear with no green fluorescence emitted.
Conclusions
Patient 70416 : Had a green fluorescence and showed signs of correlation to the positive control. The signs of the positive control and the patient were similar as anything that was positive showed the same green signs of infection. Patient 70416 therefore has signs of infection marker that was being tested.
Patient 90490 : Did not have a green fluorescence and showed signs of correlation to the negative control. The negative control no matter from what angle or any lighting, showed no signs of the infection/disease that was being tested. Patient 90490 did not have any signs of the infection marker.
BME 100 Spring 2018
