BME100 f2015:Group9 8amL5

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Contents

OUR TEAM

Leo Austin
Leo Austin
Ahmad Basiri
Ahmad Basiri
Hy Rillero
Hy Rillero
Allison Schmidli
Allison Schmidli
Michael Tyson
Michael Tyson


LAB 5 WRITE-UP

PCR Reaction Report

To set up the the reaction precise measurements of solutions had to be taken using a pipette. One member of the team did the work with the pipette while the other members observed and made sure the correct process was being followed, with the correct measurements. The pre-lab readings helped the team to understand the correct process of pipetting and how important it is to be patient and precise while using a pipette. The first stop on the pipette was used for when a sample liquid was being drawn from a sample tube into the pipette tip. The second stop on the pipette was used for when the sample liquid was released from the tip. To create the solution to be put in the PCR machine, exactly 50 microliters of PCR reaction mix was put in an empty PCR reaction tube then 50 microliters of DNA/primer mix was added, to create a combined solution of 100 microliters. There was extra liquid left in all of the DNA/Primer sample tubes, but there was no remaining PCR reaction mix leftover. For all patient and control samples of DNA and PCR reaction mix, there was not any visible amount liquid left in the sample tubes after being added to separate Buffer tubes. There was 100 microliters of PCR/DNA raction mix in each patient sample and control sample tube. The volume on the pipette was set to 120 microliters to ensure that all of the contents in those tubes were fully removed. Each sample was then added to its corresponding, pre-labeled Buffer tube. The same labeling schemed that was used for the PCR/DNA reaction mix samples was used for labeling the Buffer tubes. Because the Buffer tubes were already distinguished by having a red dot, there was no need to create a different labeling scheme.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S6
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest
    • Saturation: Highest
    • Contrast: Lowest


Camera set-up
The cradle was placed approximately 3-4 cm away from the side of the fluorimeter. The phone was placed vertically in the cradle so that the drop of solution was visible from the side with the smallest angle manageable. To get a good angle, the fluorimeter had to be raised slightly by placing it on top of a plastic tray.

  • Distance between the smart phone cradle and drop = 8 cm


Placing Samples onto the Fluorimeter

  1. Set the pipette to 80 micro liters
  2. Put on clean tip
  3. Place a clean slide in the fluorimeter
  4. Using the pipette, place an 80 microliter drop of SYBR GREEN I in the middle of the first two rows on the slide
  5. Dispose of pipette tip, and replace with a new clean tip
  6. Use the pipette to get 80 microliters of one of the calf thymus solutions
  7. Add the 80 microliters to the SYBR GREEN I already on the slide
  8. Adjust the slide so that the LED light is aligned with the drop on the slide
  9. After the solution is photographed, remove solution from the slide using the pipette
  10. Dispose of pipette tip
  11. Move the slide so that the light goes through the next two rows, so as to avoid cross-contamination. Dispose of slide if there are no more unused row available
  12. Repeat for each following sample


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL sample:

0.5 μg/mL sample:

Zero DNA sample:

Calibrator Mean Values


Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL) Final DNA Conentration in SYBR Green I Solution (microgram/mL) Sample Number RAWINTEDROP- Background ' ' Mean Standard Deviation
Image 1Image 2Image 3
52.5C-11506799146592415446761505799.66739385.50973
21C-2154101714241871503929148971159698.62534
10.5C-3127548812162631237750124316729981.79569
0.50.25C-4792094746958736393758481.666729584.56558
0.250.125C-5677825714341641428677864.666736456.51618
00C-6392497372321417441394086.333322601.94871


Calibration curves


Images of Our PCR Negative and Positive Controls

Positive Control:

Negative Control:


PCR Results: PCR concentrations solved

PCR Product Tube Label Mean of RAWINTDEN DROP-BACKGROUND PCR Product Concentration (micrograms/mL) Total Dilution Initial PCR Product Concentration (micrograms/mL)
Positive Control11308675.720.0212240.24
Negative Control20540692.871234.44
Tube 1-111396440.720.1812242.16
Tube 1-211701370.720.7512249
Tube 1-311274214.319.9512239.4
Tube 2-1864547715.9312191.16
Tube 2-210531844.718.5812222.96
Tube 2-31145064920.2812243.36


PCR Results: Summary

  • Our positive control PCR result was 240.24 μg/mL
  • Our negative control PCR result was 34.44 μg/mL


Observed results

  • Patient 12307 :

The droplets in the images for this patient were green, and the fluorescence was clearly visible. The three test tubes for this patient gave an average initial PCR Product concentration of 243.52 μg/mL.

  • Patient 19722 :

The droplets in the images for this patient were green, and the fluorescence was clearly visible. The three test tubes for this patient gave an average initial PCR Product concentration of 219.16 μg/mL.


Conclusions

  • Patient 12307 :

Because this patient's average initial PCR Product concentration of 243.52 μg/mL was significantly closer to the initial PCR Product concentration of the positive control, we concluded that this patient was positive.

  • Patient 19722 :

Because this patient's average initial PCR Product concentration of 219.16 μg/mL was significantly closer to the initial PCR Product concentration of the positive control, we concluded that this patient was positive.



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