BME100 f2014:Group29 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Amanda Smith
Name: Blake Morrow
Name: Julian Bertrandt
Name: Gergey Mousa
Name: Mitchell Dublin
Name: Zied Alghamdi


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: High
    • Saturation: High
    • Contrast: Low


Calibration
First, place a slide into the fluorimeter and turn the camera on. Next, place the camera in the cradle and make sure the camera is aligned with the surface of the slide. You may need to raise or lower the camera or fluorimeter. Once the camera is properly aligned, make sure there is an ample amount of room to slide the box over the camera and fluorimeter so that it will be completely dark when the picture is taken. Finally, measure the distance between the camera and the fluorimeter.

  • Distance between the smart phone cradle and drop = 5cm


Solutions Used for Calibration

Initial DNA Concentration(µg/mL) Volume of DNA solution (μL) Volume of SYBER GREEN (μL) Final DNA Concentration (μg/mL)
5 80 80 2.5
2 80 80 1
1 80 80 0.5
0.5 80 80 0.25
0.25 80 80 0.125
0 80 80 0




Placing Samples onto the Fluorimeter

  1. Mix all solutions in their designated test tubes
  2. Set the micro-pipette to the desired volume
  3. Using and clean tip, push and hold the button to the first stop, place the tip into the solution, and then release the button
  4. Align the pipette in the middle of the first two dots and slowly push the button all the way down, being careful not to splash the solution
  5. Throw the used tip in the waste bucket
  6. Repeat each step for each solution, using a clean tip each time and moving the slide in order to use a clean section of it for each trial


Data Analysis

Representative Images of Negative and Positive Samples

Positive Image

Postive1.jpg

Negative Image

Negative.jpg


Image J Values for All Calibrator Samples


Final DNA concentration in SYBR Green I solution (µg/mL) Area Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN DROP -- BACKGROUND
0(1) 81440 182.157 14834828 349820 14485008
0(2) 47044 216.611 10190270 228061 9962209
0(3) 47504 211.017 10024167 567097 9457070
.125(1) 40392 188.574 4789460 135365 4654095
.125(2) 30552 177.525 3590621 117614 3473007
.125(3) 40392 110.811 4475860 131342 4344518
.25(1) 44228 143.841 6361796 139242 6222554
.25(2) 42304 149.935 6342866 139684 6203182
.25(3) 34376 153.54 5278078 123830 5154248
.5(1) 42752 140.039 5986927 129819 5857108
.5(2) 40392 147.53 5959021 122723 5836298
.5(3) 45240 151.622 6859357 153690 6705667
1(1) 42752 194.347 8308710 131135 8177575
1(2) 45116 195.155 8804618 141223 8663395
1(3) 42304 207.948 8797027 148308 8648719
2.5(1) 40392 219.747 8876005 140522 8735483
2.5(2) 49884 208.879 10419715 173542 10246173
2.5(3) 51572 222.291 11463994 180723 11283271


Final DNA concentration in SYBR Green I solution (µg/mL) RAWINTDEN DROP -- BACKGROUND ' ' MEAN STANDARD DEVIATION
1 2 3
0 14485008 9962209 9457070 11301429 2768604.845
0.125 4654095 4373007 4344518 4457206.667 171104.2595
0.25 6222554 6203182 5154248 5859994.667 611271.2873
.5 5857108 5836298 6705667 6133024.333 496032.2387
1 8177575 8663395 8648719 8496563 276349.153
2.5 8735483 10246173 11283271 10088309 1281209.079


Calibration curve

DNA Concentration.jpg


PCR Results Summary

  • Our positive control PCR result was 0.18453 μg/mL
  • Our negative control PCR result was 0.44294 μg/mL

Observed results

  • Patient 39635 : the image turned out to be a rounded beach ball shape, the coloring seemed to be black and white, but was brighter than the (red) and (blue) filters that were given, 0.18453 μg/mL.
  • Patient 64133 : the image was similar to Patient 39635, the shape shape was given and the coloring was the same, 0.44294 μg/mL.

Conclusions

  • Patient 39635 : was positive, the three initial concentrations of 1-1, 1-2, 1-3 were closest to the observed positive control.
  • Patient 64133 : was inconclusive, the three initial concentrations of 2-1, 2-2, 2-3 were inconclusive because one resulted in a negative initial concentration, one yielded a positive result and one a negative.




SNP Information & Primer Design

Background: About the Disease SNP

A nucleotide is one of the core elements of DNA and RNA, consisting of a base chemical and a molecule of sugar and phosphoric acid. A polymorphism is a variation that exists inside DNA nucleotide where one of the bases has been substituted that may occur when cells replicate. The species that this SNP is found is Homo sapiens, specifically located on the chromosome 21, position on the chromosome is 34370656

The significance of this SNP is that it is likely pathogenic, meaning it probably can cause disease or other harm to an organism. The SNP rs16991654 is associated with KCNE2 gene. The official name for that gene is “potassium voltage-gated channel, Isk-related family, member 2” (genetics home reference). The function based on the name is the control of potassium as it interacts with cells. Channels that are composed with KCNE2 are present in the heart muscle, which is apart of the function that is in charge of recharging that cardiac muscle to continue a regular rhythm. Other functions of the gene include regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume (NCBI).

Disease linked to SNP is the congenital long QT syndrome that can cause irregular and spastic heartbeats. An allele is an alternative gene form that may arise through mutation and are located at the same place on a chromosome. in the SNP the disease-associated allele is the CTC sequence rather than what it should contain the sequence TTC. In the Gene the numerical position of the SNP is at 21:34370656

Primer Design and Testing

Primer without Disease

The results below with the two primers is Chr 21:35742936+35743155. This a chromosome where the rs16991654 SNP is found and the primer test is in a human genome without the TQ disease.

Forward Primer:C A T G G T G A T G A T T G G A A T G T

Reverse Primer:C C C T T A T C A G G G G G A C A T T T

USCS Picture.jpg

Primer with Disease

With these results were made from changing the final base of TTC to CTC on the primer without disease. Results were not found because the primer test does not contain the allele that is associated with the variation of the TTC.

Forward Primer: C A T G G T G A T G A T T G G A A T G C

Reverse Primer: C C C T T A T C A G G G G G A C A T T T

USCS Picture 2.jpg