BME100 f2017:Group9 W1030 L5

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OUR TEAM

Name: Nicholas Lee
Name: Cassity Jones
Name: Carlos Ruiz
Name: Samantha Balconi
Name: Rachel Oberlander


LAB 5 WRITE-UP

PCR Reaction Report

Our group felt well prepared while micropipetting and the pre-lab readings and videos gave us all a proper understanding of how to use the materials. Additionally, a member of our group had experience with micropipettes and was able to provide insight and advice while we performed the lab. We understood the difference between the first and second stop and we were able to use them with relative ease. The final reaction retained the same amount of liquid because we used the pipette to extract all of the liquid in the samples that we had. There was no liquid left in the tubes of DNA samples or the PCR reaction mix. We did not change the label scheme, because it was familiar to us and we saw that it was the most efficient.

Fluorimeter Procedure

Imaging set-up
The first part of the setup was to ensure that the proper PPE was put on. Then the following materials were gathered. A fluorimeter, 3 glass slides, micropipette, pipette holding box, iPhone 6(s). The camera was first set up to find the proper distance and height of the fluorimeter. The iphone was placed into the phone stand and was placed 7.5cm away from the fluorimeter. The fluorimeters height was adjusted by stacking small white boxes underneath it. The height was made so that the camera was directly aligned with the glass slide and had an optimal view of the beam of light. The fluorimeter was turned on and a slide was inserted so that the beam of light crossed directly through two of the circles on the slide. The phone was set on a timer of 3 seconds to take an image, this would occur three times for each sample. The phone was focused on the sample in the slide and the fluorimeter cover was placed on the whole set up. The images were taken while the cover was on and the sample was in darkness.

Placing Samples onto the Fluorimeter

  1. Insert slide into fluorimeter with the rough siding facing upward
  2. The micropipette dial was set to 80 µL
  3. Extract the SYBR Green I
  4. Place the SYBR Green I on the slide in between the two circles where the beam of light shines across
  5. Repeat steps 3 and 4 for the sample
  6. place the sample on top of the SYBR Green I


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA


High Calf Thymus DNA Image

G9 5 Picture.png


Low Calf Thymus DNA Image

G9 .5 Picture.png


Zero Calf Thymus DNA Image

G9 0 Picture.png

Calibrator Mean Values


Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND (Image 1) RAWINTDEN DROP - BACKGROUND (Image 2) RAWINTDEN DROP - BACKGROUND (Image 3) MEAN Standard Deviation
5 2.5 C-1 219957 222535 231709 224734 6176.80964
2 1 C-2 237898 251665 263075 250879 12606.87457
1 0.5 C-3 248869 435608 261913 315463 104252.5404
0.5 0.25 C-4 396062 239558 273599 303073 82309.85974
0.25 0.125 C-5 242373 320502 292651 285175 39597.34024
0 0 C-6 387280 251162 311667 316703 68198.59568


Calibration curves

G910 Dot Plot 1.png

G910 Dot Plot 2.png

Images of Our PCR Negative and Positive Controls


PCR Negative Control Image

G9 Negative Picture.png


PCR Positive Control Image

G9 Positive Picture.png


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP-BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration

(µg /mL)

G9+ 8454332 -461.735 12 -5540.823
G911 11865515.6 -655.091 12 -7861.093
G912 15228216.6 -845.699 12 -10148.385
G913 21831970 -1220.019 12 -14640.225
G9- 4732420.66 -250.766 12 -3009.197
G921 5835298 -313.281 12 -3759.369
G922 4973007.33 -264.404 12 -3172.843
G923 4122233.66 -216.179 12 -2594.151


PCR Results: Summary

  • Our positive control PCR result was -55540.823 μg/mL
  • Our negative control PCR result was -3009.197 μg/mL


Observed results

  • Patient 54046 : glowing green, -10883.234
  • Patient 94177 : not glowing green, -3175.454


Conclusions

  • Patient 54046 : negative
  • Patient 94177 : positive