BME100 f2014:Group29 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

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

Image:Postive1.jpg

Negative Image

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)81440182.1571483482834982014485008
0(2)47044216.611101902702280619962209
0(3)47504211.017100241675670979457070
.125(1)40392188.57447894601353654654095
.125(2)30552177.52535906211176143473007
.125(3)40392110.81144758601313424344518
.25(1)44228143.84163617961392426222554
.25(2)42304149.93563428661396846203182
.25(3)34376153.5452780781238305154248
.5(1)42752140.03959869271298195857108
.5(2)40392147.5359590211227235836298
.5(3)45240151.62268593571536906705667
1(1)42752194.34783087101311358177575
1(2)45116195.15588046181412238663395
1(3)42304207.94887970271483088648719
2.5(1)40392219.74788760051405228735483
2.5(2)49884208.8791041971517354210246173
2.5(3)51572222.2911146399418072311283271


Final DNA concentration in SYBR Green I solution (µg/mL) RAWINTDEN DROP -- BACKGROUND ' ' MEAN STANDARD DEVIATION
123
01448500899622099457070113014292768604.845
0.1254654095437300743445184457206.667171104.2595
0.256222554620318251542485859994.667611271.2873
.55857108583629867056676133024.333496032.2387
18177575866339586487198496563276349.153
2.587354831024617311283271100883091281209.079


Calibration curve

Image: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

Image: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

Image:USCS Picture 2.jpg


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