BME100 f2014:Group28 L5

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Contents

OUR TEAM

Name: Brandt Hansen
Name: Brandt Hansen
Name: Andrew Hamidy
Name: Andrew Hamidy
Name: Kyle Henriksen
Name: Kyle Henriksen
Name: Kyle Brague
Name: Kyle Brague
Name: Mohammed Alhusayni
Name: Mohammed Alhusayni
Name: Diego Reyes
Name: Diego Reyes


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone:I pod touch fifth generation
    • Flash:Off
    • ISO setting:Standard
    • White Balance:Standard
    • Exposure:Standard
    • Saturation:Standard
    • Contrast:Standard

Calibration

  • Distance between the smart phone cradle and drop = 7cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/mL)
5 80 802.5
280801
18080.5
.58080.25
.258080.125
080800


Placing Samples onto the Fluorimeter

  1. First, make sure proper PPE is being used, this includes gloves and a lab coat.
  2. Obtain a micropipet that is able to transport 80 microliters, fluorimeter, glass slides, camera, stand for camera, and box to cover the system.
  3. Set up the system so that the camera will be able to take pictures of the side view of the sample.
  4. Place 80 microliters of the SYBER green on the rough side of the glass slide between the two dots. Then place 80 microliters of the sample being used on the orb of SYBER green.
  5. Carefully place the glass slide in the fluorimeter so the beam of light is passing through the orb, then cover the system with the box no light will be able to penetrate.
  6. Set up camera up to timer of 5 seconds, press the take picture button and close the open end of the box.
  7. Repeat steps 5 through 6 for multiple samples being tested.


Data Analysis

Representative Images of Negative and Positive Samples

Image:PositiveDNA2.jpg Image:Drop-1.png

Image J Values for All Calibrator Samples


DNA concentraion AREA Pixel count drop background drop-background mean STD
5 39576 101.092 4000822 128151 3872671 2667215 1796151
2 39576 66.07 2614781 161980 2452801 1743198 1120036
1 39576 12.744 504367 78407 425960 336244.7 185107.4
.5 39576 23.49 929623 140840 788783 619748.7 363486.2
.25 39576 19.055 754126 141578 612548 502750.7 261876.5
0 39576 5.143 203558 147809 55749 135705.3 60946.69
Label AREA Pixel count drop background drop-background mean STD Product initial
positive 24056 106.562 2782287 85221 2687066 185485 12448583.154.0262885
negative 26919 10.407 280149 82554 197595 186766 81030.43-.15849-0.01321
1-1 28646 68.8 1970856 1919927 1313904 1313904 8933002.0801930.173349
1-2 22392 106.919 2394131 95574 2298557 1596087 1067402.640656.2200
1-3 17577 101.955 1792056 60166 1731890 1194706 802615.51.84344.15362
2-1 21980 135.48 2977998 84486 2893512 1985332 13445443.41376.28448
2-224168137.493322893944923228401221526215001073.9704.3225
2-321090136.2052872568819102790658191504512966543.274159.272849

Calibration curve
Image:Chart-1-28--.png


PCR Results Summary

  • Our positive control PCR result was _3.15___ μg/mL
  • Our negative control PCR result was __-0.15__ μg/mL

Observed results

  • Patient 76447 : A value of 2.19μg/mL was obtained for our final product. It was observed to be a green when we placed the green dye in it.
  • Patient 58040 : A value of 3.55μg/mL was obtained for our final product. It was observed to be a bright green when we placed the green dye into it.

Conclusions

  • Patient 76447 : The value for this patient is higher than the negative control, but lower than the positive control. This means that it has a concentration of DNA, but that concentration is still lower than our controls concentration. This is why the color is not as bright.
  • Patient 58040 : The value for this patient is higher than both the negative and the positive control. This shows that it has a higher concentration of DNA than the positive control. This explains the bright green color of the droplet.




SNP Information & Primer Design

Background: About the Disease SNP

Single Nucleotide Polymorphisms which are frequently called snips, are the most commonly attributed genetic variation among humans. SNP's occur normall throughout a humans DNA replacing single nucleotides- the building block of DNA. These snips occur once per 300 nucleotides roughly and can act as bio markers for scientists. These snips are flags for scientits to locate where or which gene is causing a certain disease. SNP's can be used to track inheritance of diseases gnes within blood.

Primer Design and Testing

The reulsts of our primer test were conclusive. The non-disease primers or the unaltered section, the test proved to be successful of a match. When we tested the disease primer the test was a fail which means that the two sections where not a match. This is because of the single replacement of a nucleotide in the diseased primer.

Image:DNA1-1.png

Image:DNA1-2.png

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