BME100 f2015:Group12 8amL5

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Contents

OUR TEAM

Name: Krystal Schmitt
Name: Krystal Schmitt
Name: Ladan Kamali Sarvestani
Name: Ladan Kamali Sarvestani
Name: Nathan McAdams
Name: Nathan McAdams
Name: Ashwin Krishnadoss
Name: Ashwin Krishnadoss
Name: Fangchi Shao
Name: Fangchi Shao
Name: Matthew Levins
Name: Matthew Levins


LAB 5 WRITE-UP

PCR Reaction Report

In today's Lab, we used micropipets to transfers samples into test tubes. Our prelab this week went over how to properly use the micropipets, specifically with differentiating between the first stop and the second stop on the pipet and how to utilize them (first stop for pulling up sample and second stop for discarding sample.) The prelab was extremely helpful for the students who had no prior experience with the micropipets and it was also a good refresher for those who hadn't used them in a while. Conducting the lab was very successful; however, there was one instant where some fluid got sucked back up into the pipet after discharge and we were unable to get the rest out (a very little amount.)

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung edge
    • Flash: Off
    • ISO setting: N/A
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Camera set-up

  • Distance between the smart phone cradle and drop = 15cm


Placing Samples onto the Fluorimeter

  1. First, place a 160 microliter drop of H20 on a slide and place it in the fluorimeter with the black box covering the apparatus and the blue beam shining through the drop
  2. Using a smart phone, capture a profile picture of the drop (making sure the set up was good enough to continue)
  3. Place 80 microliters of SYBR green solution on a slide and add 80 microliters of the 5 micrograms/mL of calf thymus solution
  4. Take 3 separate pictures of this drop in the black box with the blue beam shining through it
  5. Next, discard the solution from the slide and adjusted it for use of a new drop (or grab an entire new slide if necessary)
  6. We repeated steps 3-5 for 2, 1, 0.5, and 0.25 micrograms/mL of calf thymus solution
  7. Next, transfer 100 microliters of each patient sample (PCR from last lab) into a buffer solution tube (using a difference tube for each PCR sample) and invert them to mix solutions
  8. Using a slide, transfer 80 microliters of SYBR green and 80 microliters of each buffer solution (do each one separately)
  9. Take 3 pictures of the drops created on the slides for each SYBR green, PCR mixture



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA
Image:5g.png
Image:0.5.png
Image:Water.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 ' ' MEAN Standard Deviation
Image 1Image 2Image 3
0.50.25C-136672290372829083824638.77063
0.250.125C-231743318793052231381.33747.3047125
10.5C-338563389023174836404.334036.063718
21C-440395399243553038616.332683.197781
52.5C-548724426334288944748.673445.118334
00C-600000



Calibration curves
Image:KS.jpg


Images of Our PCR Negative and Positive Controls
Image:First one.png
Image:Fifth.png

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)
2661320167.3764684122008.51762
y2675348168.26241122019.14892
#2298222144.4449286121733.339144
 ?2654999166.9772641122003.727169
+2715039170.7691045122049.229254
x2128944133.7541367121605.04964
*2155289135.4179613121625.015536
 !2121078133.2573576121599.088291



PCR Results: Summary

  • Our positive control PCR result was 170.7691045 μg/mL
  • Our negative control PCR result was 167.3764684 μg/mL


Observed results

  • Patient 38292 : The image of Patient 38292 appeared to be a drop of water with a green tint. The three trials we conducted produced values of 133.7541367μg/mL, 135.4179613μg/mL, and 133.2573576μg/mL.The mean of the three trials for this patient was 135.1431519μg/mL.
  • Patient 15049 : The image of Patient 15049 appeared to be a drop of water with a slight green tint. The three trials we conducted produced values of 144.4449286μg/mL, 166.9772641μg/mL, and 170.7691045μg/mL. With an average value of 160.7304324μg/mL.


Conclusions

  • Patient 38292 : The positive control PCR result was 170.7691045μg/mL. The mean value of the trials for this patient was 135.1431519μg/mL. For this reason, it can be concluded that this patient was negative because of how significantly lower the results were compared to the positive control.
  • Patient 15049 : The positive control PCR result was 170.7691045μg/mL. The mean value of the trials for this patient was 160.7304324μg/mL. For this reason, it can be concluded that this patient was negative because of the low value of the results compared to the positive control. However, this patient was much closer to the positive control compared to the other patient making it more probable that this patient could be positive compared to the other patient.



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