BME100 s2016:Group6 W1030AM L5

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OUR TEAM

Ahnaf Rahman
Aidan Jacobs
Toan Nguyen
Mukund Khanwalker
Nicholas Pederson
Parker Henderson


LAB 5 WRITE-UP

PCR Reaction Report

Our team was successful in pipetting liquids during the PCR reaction. The team was able to properly transfer the DNA samples, nucleotides, enzymes, and primers after reviewing the pre-lab reading. The first stop on the pipettor is used to collect the exact measurement of liquid indicated on the pipettor. The second stop is used to completely release all the liquid from the pipettor, ensuring that the experiment is conducted accurately.

Fluorimeter Procedure

Web camera set-up

After setting the timer on the iPhone to 3 seconds, the iPhone was laid horizontally in front of the fluorimeter. The iPhone was placed on a small platform to be able to image the drops and part of the glass slip the drops are on.


Placing Samples onto the Fluorimeter

  1. [Step 1: After placing the glass slip onto the fluorimeter with the smooth side down, 80 micro-liters were obtained of SYBR Green I solution]
  2. [Step 2: 80 micro-liters of sample/calibration solution were added to the initial 80 micro-liters of SYBR Green I solution]
  3. [Step 3: Adjust the drop in order that the blue light shines through the center of the drop]
  4. [Step 4: Cover the entire system so that it is completely dark and take 3 images on phone]


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

zero dna:

Screen Shot 2016-04-05 at 9.11.17 PM.png

.5 DNA:

Screen Shot 2016-04-05 at 9.09.35 PM.png


5 Dna

Screen Shot 2016-04-05 at 9.15.57 PM.png

Calibrator Mean Values


Capture3456.PNG


Calibration curves

Screen Shot 2016-04-05 at 9.09.38 PM.png

Screen Shot 2016-04-05 at 9.05.02 PM.png


Images of Our PCR Negative and Positive Controls

Negative

Screen Shot 2016-04-05 at 7.41.46 PM.png

Positive

Screen Shot 2016-04-05 at 7.35.35 PM.png

PCR Results: PCR concentrations solved

Screen Shot 2016-04-05 at 9.11.37 PM.png


PCR Results: Summary

  • Our positive control PCR result was 37.97402261 μg/mL
  • Our negative control PCR result was 12.70552648 μg/mL


Observed results

  • Patient 78117 (#1) :

In Patient 78117, the images show the appearance of DNA as the concentration becomes greater and the SYBR Green is more noticeable as it binds to the DNA. The PCR concentrations were diluted by 12 microLiters and had consistent results in the visibility of the DNA in the diluted solution.

  • Patient 38765 (#2) :

For Patient 38765, the images have more drastic changes in the visibility of DNA as the PCR concentrations before dilution increase by larger increments. This increase in concentration of PCR is seen in the results of the Diluted Concentrations as they have proportional results. The SYBR Green binds with the DNA structure to give it a green visible look when fluorescent light is shined through the bead of solution.

Conclusions

  • Patient 78117 (#1) :

This patient compared to the positive control value does not line up with the images and results, yet is very close to the negative control value. When observing the images, this patient not only had darker visualizations of the DNA but this correlated to the control test for the negative control value image. The results back this up in that the original non-diluted concentration of PCR is within a half of a microliter of the control value.

  • Patient 38765 (#2) :

Patient 2 is much closer to the positive control value because the concentration of DNA in the PCR Products both before dilution and after is much higher. The images representing patient 2 are closer in comparison with the images from the positive control value. Due to the increase in PCR concentration, the green light is more vibrant from the fluorescent light shining through the bead of solution. Patient 2 is closest in comparison to the values of the negative control.