Using new equipment for the first time was difficult but the pre-lab gave easy to understand instructions. It took some time for us to understand the difference between the first and second stop but by looking at the directions we were able to figure it out. The first stop takes in the liquid and the second stop releases it. Between the two stops we could feel a click within the pipette. Once we were done mixing the controls and patients tests there was a trace amount of DNA samples and PCR reaction mix left within their tubes. Because there was small varying amounts of liquid left of the DNA samples and PCR reaction mix the final reactions did not have exactly the same amount of liquid. With more practice using the pipette it would be easier to leave less amount of DNA samples and PCR reaction mix and have the same amount of liquid in the final reactions. During the experiment we did not have to change our labeling scheme, we used the same scheme that we planned to use.
Fluorimeter Procedure
Imaging set-up
The first step was placing the slide onto the fluorimeter and the phone onto the cradle to hold it. Then the height of the flourimeter and the phone was adjusted so that the phone would be able to just see above the edge of the slide. 80 microliters of the Green 1 solution was added to the first section of the slide, followed by 80 microliters of the calibration solution. The slide was then adjusted so that the light from the fluorimeter was shining on the center of the 160 microliter solution. The camera was adjusted again and the distance from the fluorimeter was recorded. The lightbox was placed over the setup, and the camera was set on a timer. The camera was activated and the final flap was lowered three times to get three photos of the solution. The slide was removed and the process repeated with a new slide and new calibration solutions.
Placing Samples onto the Fluorimeter
Collect and label all necessary samples.
Add 80 microliters of SYBR Green to the rougher side of the glass slide between the first two rows. It will appear as a bubble.
Discard the pipette tip and replace it.
Add 80 microliters of one of the Calf Thyme samples to the bubble of SYBR Green.
Adjust the glass slide so that the blue LED light shines through the bubble made in step 1 and 2 so that it hits the black fiber optic fitting.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
.5 μg/mL sample
]
0 μg/mL sample
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
positive control PCR sample
negative control PCR sample
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was -1.827429919 μg/mL
Our negative control PCR result was -47.11443396 μg/mL
Observed results
Patient 14149 :
Qualitative - The image of patient 14149's sample appeared clear and showed no green color.
Quantitative - The initial PCR product concentrations of patient 14149's sample was shown to be -47.09143846μg/mL, 5.820222019μg/mL, 5.810535581μg/mL.
Patient 42390 :
Qualitative - The image of patient 42390's sample appeared clear and showed no green color.
Quantitative - The initial PCR product concentrations of patient 42390's sample was shown to be 2.500624361μg/mL, -17.13046712μg/mL, -1.465672039μg/mL.
Conclusions
Patient 14149 negative : Both the qualitative description and quantitative data showed that the sample was negative. The negative control appeared clear and had no green color which was similar to patient 14149's sample. Also the initial PCR product concentration of patient 14149's 1st sample (-47.09143846μg/mL) was similar to the negative control's concentration (-47.11443396 μg/mL). The other two samples were inconclusive.
Patient 42390 negative :Both the qualitative description and quantitative data showed that the sample was negative. The negative control appeared clear and had no green color which was similar to patient 42390's sample. Also the concentration of patient 42390's 2nd sample (-17.13046712μg/mL) was closest to the initial PCR product concentration of the negative control's concentration (-47.11443396 μg/mL). While the third sample's initial PCR product concentration (-1.465672039μg/mL) was similar to the positive's (-1.827429919 μg/mL) the visual aspect pushes more for a nagative sample.
BME 100 Spring 2017
