BME100 s2016:Group16 W1030AM L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Mayar Allam
Role(s)
Name: Tashena Jackson
Role(s)
Name: Ryanne Maxie
Role(s)
Name: Andrew Smith
Role(s)
Name: Joseph Carrillo
Role(s)
Name: Austin Morgan
Role(s)


LAB 5 WRITE-UP

PCR Reaction Report

Our team's experiece with pipetting the samples (DNA, primers, necleotides, and enzymes) to set up the reaction was successful. The pre-lab reading did help a lot. We made sure we understood the difference between the first and second stop on the pipettor. The first stop of the pipettor measures the exact amount we set the device to. We made sure that the tip was half way submerged in the liquid and paused for one second before we removed the pipette. The second stop on the pipettor releases all the liquid that was draw into the tip. We also paused for two seconds before released the plunger.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone 6S
  • Flash: no
  • ISO: 800
  • White Balance: Auto
  • Exposure: Auto
  • Saturation: Auto
  • Contrast: Auto

Camera set-up

For our documentation records we used an iPhone 6s. Before setting the iPhone camera in place, we adjusted the timer to three seconds so that we would have time to focus and black out the surroundings before the picture was taken. Then we placed the iPhone camera four centimeters away from the fluorimeter on a slightly elevated stand so that the camera was level and perpendicular with the droplette. The fluorimeter and iPhone camera are placed inside the lightbox when the picture is ready to be taken.


Placing Samples onto the Fluorimeter

  1. Detect the smooth side of the slide
  2. Place the smooth side face down on the Fluorimeter
  3. Calibrate the micropipetter so that it is set for 80 μL, then attach a clean new tip
  4. Between the first middle two circles carefully place 80 μL of SYBR Green I Solution
  5. Discard of previously used pipette tip and replace with a new one
  6. Add 80 μL of the sample or blank to the SYBR Green I drop
  7. Adjust the slide so that the blue light goes straight through the center of the sample
  8. Place a black box over the fluorimeter that that all light is blocked out
  9. Adjust Camera so that it is able to capture the sample
  10. Set a timer on the Camera to 3 seconds
  11. Push capture on the Camera and then close the box
  12. Once this is done repeat the process until 3 images are captured
  13. Remove the slide and clean off previous materials
  14. Repeat all steps for all remaining samples



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA High Concentration (5 μg/mL sample)
High.PNG
Low Concentration (.5 μg/mL sample)
Low.PNG
No Concentration
No Concentration.PNG


Calibrator Mean Values


BME100Cal.PNG

Calibration curves
Dot plot 1 .png
Dot Plot 2 .png

Images of Our PCR Negative and Positive Controls

G16 Positive
G16 Positive.png
G16 Negative
G16 Negative.png


PCR Results: PCR concentrations solved

Final Results.PNG


PCR Results: Summary

  • Our positive control PCR result was 132.256 μg/mL
  • Our negative control PCR result was -160.433 μg/mL


Observed results

  • Patient 29688: The PCR concentration for this patient was on average -62 micrograms per milliliter, which puts it closer to the negative control value than the positive control, although it is still fairly far from our negative control.
  • Patient 65775 : The PCR concentration for this patient was on average, and excluding the outlier, 372 micrograms per milliliter, which is about twice the calculated value for our positive control.


Conclusions

  • Patient 29688:

The values of the initial PCR product concentration for the first, and the second sample were closer to the negative control initial PCR product concentration. However, the value of the initial PCR product of the third sample was closer to the value of positive control. As a result, It was assumed that patient 29688 should be considered negative. This value however, and our value for the negative control too are both fairly unreasonable, as it is not possible to have a negative PCR concentration. There must have been some issue in the lab proceedings that led to these poor values, in addition to our poor values for our graph which is seen by the very small R^2 value for correlation.

  • Patient 65775 :

The values of initial PCR product concentration the all three samples are all closer to the value of initial PCR product concentration of the positive control more than the negative control. Therefore, patient 65775 was considered positive.