BME100 f2015:Group1 8amL5

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Contents

OUR TEAM

Name: Tiffany Nguyen
Name: Tiffany Nguyen
Name: Maria Dooling
Name: Maria Dooling
Name: Brenden Smith
Name: Brenden Smith
Name: Nolverto Guiterrez
Name: Nolverto Guiterrez
Name: Dalia Sarbolandi
Name: Dalia Sarbolandi
Name: Nathaniel Olsen
Name: Nathaniel Olsen


LAB 5 WRITE-UP

PCR Reaction Report

The experience with micro pipetting taught us a lot. The pre-lab assignments as well as the tutorials online prepared us well for this lab. They gave us experience with how to work a micro pipetter with confidence. Furthermore, they clearly explained the differences between the first and second stop on the pipette. This allowed us to avoid inconsistencies in our data. It was particularly important in this lab to be able to correctly work a micro pipetter. Our final reactions had an equal amount of liquid so there was not remaining liquid in the DNA and PCR mixture. The tubes were labeled with marker. The tutorials and pre lab materials helped accomplish this. All in all, much was learned about the process of micro pipetting and how to use a fluorimeter.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Iphone 5 and Iphone 6
    • Flash: Off
    • ISO setting: NA
    • White Balance: NA
    • Exposure: High
    • Saturation: High
    • Contrast: Low


Camera set-up

  • Our camera was placed into the cradle and lifted
  • Distance between the smart phone cradle and drop = 5.5 cm


Placing Samples onto the Fluorimeter/Taking Fluorimeter Measurements

  1. Put on gloves and find the smooth side of the glass slide
  2. Turn on the fluorimeter
  3. Place slide in the fluorimeter smooth side down
  4. Set cellphone camera for 3 seconds and place in the cradle
  5. Adjust height of fluorimeter so that the camera will take a picture of the slide edge-on
  6. Place an 80uL drop of the SYBR Green I solution on the first two clear circles in the middle of the slide
  7. Place an 80uL drop of the sample/calibration solution directly on top of the SYBR Green I solution
  8. Move the slide around until the light hits the drop and focuses the light on the other side
  9. Adjust the distance between the smartphone and the fluorimeter so that is is greater than 4cm away but not blurry. Record set distance.
  10. Cover with lightbox and check that the camera is focused
  11. Press camera button and lower flap before picture it taken
  12. Discard liquid drop
  13. Move slide to the next position
  14. Repeat steps 1-14 with all 5 possible measurement positions.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High Calf Thymus DNA

Low Calf Thymus DNA

Zero Calf Thymus DNA

Calibrator Mean Values


Initial Concentration of 2x Calf Thymus DNA solution (µg/mL) Final DNA Concentration in SYBR Green I solution (µg/mL) Sample # RAWINTDEN Drop - Background Mean Std Dev
Img 1 Img 2 Img 3
5 2.5 G-1 18678998 17876541 19128456 18561332 634198
2.5 1.25 G-2 12562821 13982346 11287635 12610934 13480000
1 .5 G-3 11783472 10236814 9126734 10382340 1334334
.5 .25 G-4 8345724 8178942 7976254 8166973 185026
.25 .125 G-5 5422637 6134876 5798234 5785249 356297
0 0 G-6 2634578 2390456 2623987 2549674 137988


Calibration curves


Images of Our PCR Negative and Positive Controls

Postive

Negative


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) "PCR Product Concentration (µg /mL)
(Step 5 calculation)"Total Dilution"Initial PCR Product Concentration
(µg /mL)
(Step 6 calculation)"
G1+92548721.21341214.5608
G1-43258255.57721266.9264
G1 1-139245735.88291270.5948
G1 1-241827365.76211269.1452
G1 1-346235495.27341263.2808
G1 1-437892516.24731274.9676
G1 1-544897455.49051265.886
G1 1-648723694.92351259.082


PCR Results: Summary

  • Our positive control PCR result was 14.5608 μg/mL
  • Our negative control PCR result was 66.9264 μg/mL


Observed results

  • Patient 31801: After combining the SYBR green and the patient's PCR sample, it was evident that the patient was negative. No visible sign of green was detected through the fluorimeter or ImageJ.
  • Patient 21190 : The second patient also came up as negative after the test. The fluorimeter and ImageJ did not detect any sign of green within the sample.


Conclusions

  • Patient 31801 : Negative
  • Patient 21190 : Negative

Based on the results of the PCR product concentrations, we found that patients 31801 and patient 21190 were both negative for the disease. No green appeared in the pictures from the fluorimter in either patient so this is how the conclusion was reached that both patients were negative.


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