BME100 f2014:Group23 L5

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Contents

OUR TEAM

Name: Cesar Marin
Name: Cesar Marin
Name: Brady Dennison
Name: Brady Dennison
Name: Kassandra Flores
Name: Kassandra Flores
Name: Danielle Beach
Name: Danielle Beach
Name: Theodore Kyriacou
Name: Theodore Kyriacou
Name:  Joshua Kahn
Name: Joshua Kahn


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy Note 3
    • Flash: None
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: Highest Setting
    • Contrast: Highest Setting


Calibration

The smartphone was placed in the cradle with the camera app on. The cradle was then moved so that the phone was 12 cm away from from where the drop would be measured. Pieces of bubble wrap were placed in between the phone and the cradle so that the phone was level with the drop.

  • Distance between the smart phone cradle and drop = 12 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL) Volume of 2X DNA Solution (µL) Volume of SYBR Green I Dye Solution (µL) Final DNA Concentration in SYBR Green I Solution (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

Solutions Used for Measuring PCR Products

PCR Product Tube Label Volume of the Diluted PCR Product solution(μL) Volume of SYBR Green I Dye Solution (µL) Dilution 1 Dilution 2 Total Dilution Simplified Fraction
G23 + 80 80 0.166 0.5 0.0833
G23 - 80 80 0.166 0.5 0.0833
G23 1-1 80 80 0.166 0.5 0.0833
G23 1-2 80 80 0.166 0.5 0.0833
G23 1-3 80 80 0.166 0.5 0.0833
G23 2-1 80 80 0.166 0.5 0.0833
G23 2-2 80 80 0.166 0.5 0.0833
G23 2-3 80 80 0.166 0.5 0.0833


Placing Samples onto the Fluorimeter

  1. Line up fluorimeter light with the section on slide where the sample will be placed (middle of two circles)
  2. Put new tip into micropipette and collect 80uL of SYBR green
  3. Place 80 uL of SYBR green into area from step 1, lined with the light
  4. Discard tip, get new tip and collect 80uL of desired sample
  5. Place 80uL of sample on top of SYBR green in the fluorimeter slide
  6. Adjust slide on fluorimeter to allow for the narrowest possible stream of light
  7. Place camera on a timer
  8. Place camera and fluorimeter in a box to block out light, and wait until picture is taken


Data Analysis

Representative Images of Negative and Positive Samples

Sample With No DNA

Image:No DNA.png

Sample With DNA

Image:DNAA.png

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#111728162.94673823241201757262149
0#29594455.8675360083827615277322
0#310248056.23257626761220175640659
0.125#120096758.1291168208023088111451199
0.125#220939761.1561280586231602812489834
0.125#322028159.2431305012831544112734687
0.25#1148276105.6841567036516686015503505
0.25#2143862104.8521508423716857314915664
0.25#3148848104.2261551380417677915337025
0.5#163004121.8327675924846897591235
0.5#253899121.3866542599670566475543
0.5#363250120.0757594743714777523266
1#1101788159.5631624162011110016130520
1#2107815154.6561667422412157016552654
1#3106365155.6671655750711604016441467
2.5#152043224.1161166364727260711391040
2.5#296387201.4551941768633967919078007
2.5#393996203.5541913322048949418643726
PCR Product Tube Label Area Mean Pixel Value RAWINTDEN of the Drop RAWINTDEN of the Background RAWINTDEN Drop - Background
G23 + #181288211.8731722273951930416703435
G23 + #283298209.7431747121047736416993846
G23 + #384916206.9821757609338035317195740
G23 - #16326247.93530324951141822918313
G23 - #26536452.55134349641261523308812
G23 - #37304853.77239279591847993743160
G23 1-1 #163236217.1351373075629177213438984
G23 1-1 #269676214.2281492655527465914651896
G23 1-1 #372935209.6911529378648453314809253
G23 1-2 #168930223.9181543463574805414686581
G23 1-2 #283892214.711801246254281817469644
G23 1-2 #378844213.2531681370655236116261345
G23 1-3 #179201218.2611728650565301616633489
G23 1-3 #291457222.25720326925100785219319073
G23 1-3 #377372225.7381746583869735716768481
G23 2-1 #186508221.8761919408984924318344846
G23 2-1 #279512230.2361830653671724917589287
G23 2-1 #384287228.6991927632870850018567828
G23 2-2 #177942224.5411750120426407417237130
G23 2-2 #266272229.6131521688126039614956485
G23 2-2 #369675229.8641601575725040915765348
G23 2-3 #146398225.5991046735227137210195980
G23 2-3 #239267223.42487731802188588554322
G23 2-3 #354020228.3651233629343654511899748
Final DNA concentration in SYBR Green I solution (µg/mL) RAWINTDEN DROP - BACKGROUND ' ' ' '
123MeanStandard Deviation
07262149527732256406596060043.3331056786.151
0.12511451199124898341273468712225240681427.0689
0.2515503505149156641533702515252064.67302990.0229
0.57591235647554375232667196681.333625448.0938
116130520165526541644146716374880.335044544.086
2.511391040190780071864372616370924.334318169.285
PCR Product Tube Label RAWINTDEN DROP - BACKGROUND ' ' '
123Mean
G23 +16703435169938461719574016964340.33
G23 -2918313330881237431603323428.333
G23 1-113438984146518961480925314300044.33
G23 1-214686581174696441626134516139190
G23 1-316633489193190731676848117573681
G23 2-118344846175892871856782818167320.33
G23 2-217237130149564851576534815986321
G23 2-31019598085543221189974810216683.33

Calibration curve
Image:Final_DNA_SYBR.png

PCR patient label Mean RAWINTDEN drop-background PCR product concentration Initial PCR product concentration
G23 +16964340.33 2.32 ug/mL 27.84 ug/mL
G23 -3323428.333 -2.23 ug/mL -26.76 ug/mL
G23 1-114300044.33 1.43 ug/mL 17.16 ug/mL
G23 1-216139190 2.05 ug/mL 24.6 ug/mL
G23 1-317573681 2.52 ug/mL30.24 ug/mL
G23 2-1 18167320.33 2.72 ug/mL 32.64 ug/mL
G23 2-2 15986321 1.99 ug/mL 23.88 ug/mL
G23 2-3 10216683.33 .072 ug/mL 0.864 ug/mL

PCR Results Summary

  • Our positive control PCR result was 27.84 μg/mL
  • Our negative control PCR result was -26.76 μg/mL

Observed results

  • Patient 1-33849 : All of the images for patient 1 were bright green. The green was mostly at the bottom, with blue at the top. Patient 1 had an average initial PCR product concentration of 24 μg/mL.
  • Patient 2-19967 : All of the images for patient 2 were also bright green. The green fluorescence was shown all throughout the drop. Patient 2 had an average initial PCR product concentration of 18.869 μg/mL.

Conclusions

  • Patient 1-33849 : Patient 1 had an average initial PCR product concentration that was 3.84 μg/mL less than that of the product concentration of the positive sample, and 50.76 μg/mL greater than that the product concentration of the negative sample. Since Patient 1 had a concentration closer to the concentration of the positive sample, Patient 1 is positive. All of Patient 1's initial concentrations were closer to the concentration of the positive sample than the negative sample.
  • Patient 2-19967 : Patient 2 had an average initial PCR product concentration that was 8.9712 μg/mL less than that of the product concentration of the positive sample, and 45.6288 μg/mL greater than that the product concentration of the negative sample. Since Patient 2 had a concentration closer to the concentration of the positive sample, Patient 2 is positive. All of Patient 2's initial concentrations were closer to the concentration of the positive sample than the negative sample. Positive initial concentrations suggest that the patient is positive, while negative initial concentrations suggest that the patient is negative.




SNP Information & Primer Design

Background: About the Disease SNP

A nucleotide is the basic building block of DNA and other nucleic acids. the four main nucleotides are A,C,T,and G. A polymorphism is a common change in DNA sequences that appears in the phenotypes of different groups of population. The SNP rs16991654 variation is found in homosapiens. This variation is found in the 21:34370656 chromosome. The clinical significance is this SNP is that it is pathogenic. This SNP is associated with the KCNE2 gene. This SNP is linked to the disease of congenital long QT syndromes. KCNE2 stands for potassium voltage-gated channel, Isk-related family, member 2. The molecular function of this gene includes regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele in an alternative to a gene sequence that are done through mutation of a nucleotide base pair. The disease-associated allele contains the sequence CTC. The numerical position of the SNP is 34,370,656. The non-disease forward primer is catggtgatgattggaatgt. The numerical position exactly 200 bases to the right of the disease SNP is 3,430,856. The non-disease reverse primer is cccttatcagggggacattt. The diseases forward primer is catggtgatgattggaatgc. The disease reverse primer is cccttatcagggggacattt.


Primer Design and Testing

When the non-disease forward primer was tested, it was found in the database. This is because a part of the DNA that was selected did not contain the sequence that causes the disease. The disease specific primers did not exist because one nucleotide was changed in the DNA sequence. While this may cause the disease, it affects the entire sequence, it does not exist since it did not change by a group of three nucleotides, or an entire codon.

Non-Disease Forward Primer Results

Image:Part a.png

Disease Specific Primers Results

Image:Part b.png


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