BME100 f2016:Group6 W1030AM L5

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Biomarvel Engineroes

Name: Matt Edick
Name:  Edgar Manriquez
Name:  D. Brennen Martin
Name: Megan O'Reilly
Name: Blake Stephens


LAB 5 WRITE-UP

PCR Reaction Report

To set up the reaction, one member of the group pipetted 80μL of a sample onto a slide. Immediately after, 80μL of SYBR Green 1 was added into the sample on the slide. This was added in order to detect the amount of DNA in the sample. This process was repeated for each sample, using a new tip with each test and with the SYBR Green 1 being exposed to as little light as possible before the images were taken. The pre-lab reading was helpful in the sense that it explained to us how the PCR reaction worked and how important it would be for our results to be accurate and careful in the pipetting process. The difference between the first and second stop on the pipettor was understood. The samples and SYBR Green 1 were sucked into the pipette until the first stop was reached, and were expelled from the pipette onto the slide by pressing down to the second stop. The second stop includes a small puff of air in order to rid all solution from the tip of the pipette, increasing accuracy of the volume of solutions.

All final reactions contained the same amount of liquid. This is was ensured by pipetting the same amount into the PCR reactions. No, there was no liquid left in the tubes after the testing. All samples were used. No, the labeling scheme did not have to be altered.

Fluorimeter Procedure

Imaging set-up

First, we calibrated our cell phone camera (HTC 10) by

                  A.   INACTIVATE THE FLASH
                  B.   SET ISO TO 800 
                  C.   SET WHITE BALANCE TO AUTO
                  D.   SET EXPOSURE TO HIGHEST SETTING
                  E.   SET SATURATION TO THE HIGHEST SETTING 
                  F.   SET CONTRAST TO LOWEST SETTING

Next, we placed the fluorimeter on top of a few containers to raise the scanning surface to be nearly level with the camera's lens (~12-13cm). Then the HTC 10 was placed vertically in a cradle about 4 cm away from the fluorimeter. A hydrophobic glass slide was placed in the fluorimeter so that the beam of light was between the first two rows of dots, and the camera was centered on the slide so that the field of vision encompasses the entire slide surface. Finally, we placed a lightbox over the entire apparatus with one side flap open, but free to close.

Placing Samples onto the Fluorimeter

  1. [Instructions: Step one, in your own words]
  2. [Instructions: Step two, in your own words]
  3. [Instructions: Step three, in your own words]
  4. [Instructions: Step etc., in your own words]


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

1. 5 μg/mL sample
File:OWW 5.jpg
2. 0.5 μg/mL
File:OWW 0.5.jpg
3. Zero DNA sample
File:OWW 0.jpg


Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (micrograms/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 33913698 32032297 34659749 33535248 1353991.989
2 1 C-2 33409685 33843845 38398996 35217508.67 2763787.25
1 0.5 C-3 30696091 31919060 33376894 31997348.33 1342115.114
0.5 0.25 C-4 25978297 25019553 28168319 26388723 1614007.112
0.25 0.125 C-5 16086778 15443798 15119831 15550135.67 492166.0286
0 0 C-6 2685407 2205939 3736224 2875856.667 782717.2874


Calibration curves

Calibration Curve 1.png
Calibration Curve 2.png


Images of Our PCR Negative and Positive Controls

1. Negative control PCR sample
File:OWW Neg.jpg
2. Positive control PCR sample
File:OWW Pos.jpg


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)
G6 Positive 18875058 0.5012926626 12 6.015511951
G6 Negative 6913972.333 -0.3412252352 12 -4.094702822
G6 1-1 2157014.667 -0.6762969907 12 -8.115563889
G6 1-2 2733622.667 -0.6356817344 12 -7.628180813
G6 1-3 -45074.667 -0.8314083015 12 -9.976899618
G6 2-1 2831914.667 -0.6287582183 12 -7.54509862
G6 2-2 2969710 -0.6190521567 12 -7.428625881
G6 2-3 2822995.667 -0.629386457 12 -7.552637484


PCR Results: Summary

  • Our positive control PCR result was 6.02 μg/mL
  • Our negative control PCR result was -4.09 μg/mL


Observed results

  • Patient 91356 : No significant fluorescence within the drop during test, and an average calculated concentration of -8.5736
  • Patient 85189 : No significant fluorescence within the drop during test, and an average calculated concentration of -7.5088


Conclusions

  • Patient 91356 : Being quantitatively closer to the value of the negative control, and due to the lack of fluorescence within the test drops (comparable to the negative control images), patient 91356 is negative on the genetic marker.
  • Patient 85189 : Being quantitatively closer to the value of the negative control, and due to the lack of fluorescence within the test drops (comparable to the negative control images), patient 85189 is negative on the genetic marker.