BME100 s2016:Group15 W1030AM L5

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OUR TEAM

Name: Sidney Covarrubias
Name: Byron Alarcon
Name: Mason Buseman
Name: Kelsey Graft
Name: Ibrahim Aljabri
Name: Jake Xu


LAB 5 WRITE-UP

PCR Reaction Report

All in all, in this experiment, Two patients submitted three samples of their DNA in order to look for a disease marker. In order to acquire an answer for either patient there was a list of clear steps that had to be followed outline in Lab 4 wiki lab report as well as the pre-lab reading. First all materials in the protocol for Lab A were acquired. After that it was necessary to cut the strip of empty PCR tubes in half to obtain to strips of four linked tubes in order to accommodate the OpenPCR machine. Now a black marker was necessary to label the sides of the empty tubes with labels specific to our group, and after that place the tubes in a tube rack We labeled all of the tubes accordingly: G151-1, G151-2, G151-3, G151-3, G152-1, G152-2, G152-3, G15P, and G15N. The labels indicate the trials for patient one (G151-1, G151-2, and G151-3), patient two (G152-1, G152-2, and G152-3), positive control (G15P), and negative control (G15N). Note: GP15 was left out of this lab report in the report following because it became redundant, twins only to signify group 15's PCR reaction tubes. At this point the experiment we began using the PCR positive control tube. Use the micropipette to transfer 50 μL of PCR reaction mix into the positive control tube. We were sure to discard the pipette tip after one use (every use) in order to avoid cross contamination. Overall, the pipetting of the samples were shaky at first but after many trials, the procedure was second nature. In order to pipette a sample using the micropipette, we had to set the sample amount (which is in microliter) press until the first stop, and place the micropipette tip within the sample, and release the stop slowly to draw in the sample. In order to release the micropipette sample, the user had to push all the way to the second stop slowly. To release the used tip press the release tab on the side of the pipette. Using a fresh pipette tip, transfer the positive control DNA/primer mix into the positive control tube. At this point the total volume in the positive control PCR reaction tube is 100 μL. The same steps will be followed for the negative control tube, patient 1 replicates 1, 2, and 3 tubes as well as patient 2 replicates 1, 2, and 3. Use the appropriate DNA/primer mix for the corresponding tubes, all of which should contain 100 μL by the end. After the process is completed for all tubes their lids should be closed tightly and they should all be taken to the assigned PCR machine. The tubes should then be placed into the slots in the heating block. Two groups will proceed with this step at the same time, so the machine can’t be started until 16 slots are filled.By the end go the lab there was still sample liquids in the test tubes, but any excess were disposed of properly and safely.

Fluorimeter Procedure

Web camera set-up
In order to set up the web camera, the group went to the Logitech website and downloaded the software. The camera was plugged into the computer through the USB and the installation was completed and the computer was restarted. The software was opened and ready to capture images. The webcam was placed on a stand 7 cm away from the drop. The webcam was raised using a phone case to get a direct image of the experiment. The fluorimeter was raised as well to get the side view.

Placing Samples onto the Fluorimeter

  1. On the first two center circles add the SYBR Green I solution in a 80 μL drop with the micropipette.
  2. Then 80 μL of the sample or calibration solution should be pipetted onto the SYBR Green I solution.
  3. Center the slide so the light goes through the drop and so that the light is focused by the drop.
  4. The webcam was adjusted until it was 7 cm away which allowed the image to be focused and close.
  5. The box given was used to shield the experiment from light but one side was lifted open.
  6. The image was checked once more to make sure that the image was focused and centered.
  7. The flap that was up was lowered carefully and the web camera software was used to take three images of the sample.
  8. Using a micropipette remove the 160 μL solution from the slide and deposit the waste into the liquid waste bin.
  9. Move the slide so that the light goes through the next two center circles.
  10. Repeat steps 1-9 until all the center circles on the slide have been used.
  11. Dispose of the slide in the sharps waste bin.
  12. Take out a new slide to continue the experiment.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High (5 μg/mL sample)

High15.png

Low (0.5 μg/mL sample)

Low15.png

Zero (0.0 μg/mL sample)

Zero15.png

Calibrator Mean Values

Table215.png

Calibration curves


DNAGraphs15.png

Images of Our PCR Negative and Positive Controls

Postive

Positive15.png

Negative

Negative15.png

PCR Results: PCR concentrations solved

Table515.png

PCR Results: Summary

  • Our positive control (+) PCR result was 26.58645908 μg/mL
  • Our negative control (-) PCR result was 1.830409507 μg/mL


Observed results

  • Patient 43290: Overall, the samples for the all three images/trials appear to be colorless, but there is a faint amount of green fluorescence. And this is reflected in the data that approximately there is a range between 1.63-6.62(μg/mL) of DNA PCR reaction concentrations.
  • Patient 40691: Overall, the samples for the all three images/trials appear to be more colorless in comparison to patient 43290, and there appears to be less or none of the green fluorescence. And this is clearly reflected in the data that approximately there is an even smaller range between 0.65-1.90(μg/mL) of DNA PCR reaction concentrations.

Conclusions

  • Patient 43290: When comparing this patient to the positive and negative results, we can conclude that this patient is more closely similar to the negative results because of the range of 1.63-6.62(μg/mL) of DNA PCR reaction concentrations. And because the 2 out of the 3 trials yielded results around the negative result of 1.83(μg/mL) of DNA PCR reaction concentrations.
  • Patient 40691: Likewise, when comparing this patient to the positive and negative results, we can conclude that this patient is more closely similar to the negative results.
  • Explanation: In retrospect, both the patients results read through the fluorimeter illustrate that these patients are closely related to the negative control which was 1.83(μg/mL). Thus, because we know that the dye used in this lab was known as SYBR Green I works and fluoresces very well in the presence of double stranded DNA and very weakly in water or with single strands of DNA. We can assume either the patient does not have the double stranded DNA that was cut and replicated from the PCR reaction, or there was an error in the procedures that yielded very few double stranded DNA and most single stranded DNA.