BME100 f2015:Group3 8amL5

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Contents

OUR TEAM

Name: Rachel Hagan
Name: Rachel Hagan
Name: David Busch
Name: David Busch
Name: Shahzadi Aimen
Name: Shahzadi Aimen
Name: Michele Dekker
Name: Michele Dekker
Name: Amellali Gamez
Name: Amellali Gamez
Name: Jose Moreno
Name: Jose Moreno


LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab helped refresh our memory on the exact procedures needed when using a micropipette. Our experience with using a micropipette was excellent and we fully understand the difference between the first and second stop of the micropipette. There was one point where there was a little bit of the liquid left on the tube, but not too bad. Overall, the use of micropipettes was success.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Note3
    • Flash: Off
    • ISO setting: Maximum
    • White Balance: Automatic
    • Exposure: 0
    • Saturation: N/A
    • Contrast: N/A


Camera set-up

  • We gathered a phone holder/stand and vertically inserted the phone. Using a plastic box we stabilized the phone. We then measured thirteen centimeters between the phone and the fluorimeter.
  • Distance between the smart phone cradle and drop = 13 centimeters


Placing Samples onto the Fluorimeter

  1. Step one: Turn on the Fluorimeter
  2. Step two: While wearing gloves place the smooth side of the slide down on the Fluorimeter
  3. Step three: Place the camera 13 centimeters away from the Fluorimeter
  4. Step four: Adjust camera to line up the picture
  5. Step five: Pipette 80 microliters of SYBR green on the slide in between the 2 sets of dots
  6. Step six: Pipette 80 microliters of the sample with the 80 microliters of SYBR green
  7. Step seven: Move the slide so the light shines through the middle of the drop

Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA


5mg/mL 5mg/mL sample


0.5 mg/mL 0.5mg/mL sample


ZERO DNA zero dna



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
52.5C-126422278302719427148.66667705.0938472
21C-228838295102951029286387.9793809
10.5C-329178270812652827595.666671397.957915
0.50.25C-432079324583150032012.33333482.4669246
0.250.125C-52945131190361421418.3333315443.50169
00C-62829129911339214891.3333312716.46572

Calibration curves
Image:Plot1group3.JPG Image:Plot2group3.JPG


Images of Our PCR Negative and Positive Controls

Positive Group positive group


Negative Group Negative Group


PCR Results: PCR concentrations solved

Image:Patientresultsgroup3.JPG


PCR Results: Summary

  • Our positive control PCR result was 45.80738602 μg/mL
  • Our negative control PCR result was 41.01719245 μg/mL


Observed results

  • Patient 33309 : The image for the positive control group was dim at first sight. After comparing to the negative control group, a slight difference in the top stripe was noticed. This was confirmed through the initial finding of the ImageJ measurement of the RAWINTDEN Of the Drop of 1058630 which was much higher than the negative control results, as discussed below. The calculation of the positive control group's PCR result was 45.80738602 μg/mL, confirming the higher concentration.
  • Patient 32924 : The image for the negative control results did show a slight stripe for the green image after analyzing the positive control group. But, the image showed noticeably less saturation than the positive control group at the measurement of RAWINTDEN of the Drop at the number 748912. The calculation of the negative control group's PCR result was 41.01719245 μg/mL, confirming a lower concentration.

While both images first appeared to have no difference between them, this was discounted through the measurements of ImageJ and the concentration levels through the calculations.


Conclusions

  • Patient 33309 : Patient one results compared to positive and negative control value was inconclusive. Further data is required to determine whether the patient one is positive or negative.
  • Patient 32924 : Patient two's results determined a positive result for the test. No further testing is necessary.



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