OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
Our experience with micropipetting the samples in this reaction were effective. We were able to successfully extract accurate amounts of microliters onto the slidess in order to be tested. The pre-lab was useful because it was able to teach our group how to efficiently pipette accurate amounts to use in our lab. We were able to understand the difference between the first and second stops while also understanding their function for pipetting. The final reactions did have the same amount of liquid as the others and there was some remaining liquid in the tubs that held the samples but that is only because we used the correct amount of microliters in our measurements. We did not have to change our labeling scheme. All we did was simply lay the PCR samples on the table in the order that we wanted to test them in and we were able to organize the data that way.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: HTC 1 M8
- Flash:No
- ISO setting:N/A
- White Balance: N/A
- Exposure:N/A
- Saturation:N/A
- Contrast:N/A
Camera set-up
A metal stand was utilized to hold the phone horizontally in place. The Phone slipped to an incorrect angle, so a spacer was created out of plastic to hold the phone at a ninety degree angle.
- Distance between the smart phone cradle and drop =
Distance in centimeters: 8.0 centimeters
For concentration 5, distance was 9.5 cm
Concentration 2, distance was 11 cm
Concentration 1, distance was 11 cm
Concentration .5, distance was 11 cm
Once we tested the DNA samples, centimeter length was 11.5
Placing Samples onto the Fluorimeter
- Step one, set the micropipettor to 80 microliters
- Step two, extract 80 microliters of the SYBR Green and place onto the slide in the fluorimeter
- Step three, Get a new tip, then extract 80 microliters of the DNA and place on the same drop of the SYBR Green
- Step four, Place phone in a proper angle in front of the fluorimeter and take a picture with the box cover closed over it
- Step five, take three pictures of each DNA sample
- Step six, clean up the sample on the slide and dispose of the tip and sample, then move the slide in the fluorimeter to a new positions
- Step seven, repeat steps listed above for each of the different DNA samples
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High photo
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Low photo
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Zero photo
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Calibrator Mean Values
TABLE GOES HERE
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive photo
Negative photo
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PCR Results: PCR concentrations solved
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PCR Results: Summary
- Our positive control PCR result was 96 μg/mL
- Our negative control PCR result was 86 μg/mL
Observed results
- Patient 97780: Because this patient was positive the SYBR Green portrayed a droplet that glowed green. The average quantitative description of this patient was about 82 (ug/mL)
- Patient 21836:Because this patient was negative, the SYBR Green droplet was a clear and transparent drop. The average quantitative description of this patient was 92.66 (ug/mL)
Conclusions
- Patient 97780: Although the image shows that it was positive due to the presence of the green color under fluorescence light, the numbers don't agree with it because the concentration was closer to the negative control than it was closer to the positive control.
- Patient 21836: Like patient 97780, this patient's value did not agree with the observation during experiment. The color showed negative result, but the values were closer to the positive than they are to the negative. This could have been a result of the calculation error that resulted in the unexpected values. Therefore, one cannot conclude that the patient was positive or negative.
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BME 100 Fall 2015
