BME100 s2015:Group9 12pmL5

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png BME 100 Spring 2015 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png


OUR TEAM

Name: Brianna Chavez
Name: Johnathon Ruiz
Name: Josh Quick
Name: Maria Guzman
Name: Taylor Little
  • In US, lab group #1


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 6 Plus
    • Flash: None
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest
    • Saturation: Lowest
    • Contrast: Highest


Calibration
After setting up the black box and putting the fluorimeter inside the black box close to the back, and turning it on once it is in place. Next get your phone and make sure there is no blur or technical problems then go set up the camera with a timer of 3 seconds and then place it on the cradle.Make sure to get a ruler with centimeters and the phone cradle. Then put down the cradle in front of the fluorimeter facing its side and place the phone in the cradle to accurately measure the distance needed. Measure 4 cm from both sides of the cradle in respect of the phone's position to have a close to precise measurement of distance and and turn it on by.

  • Distance between the smart phone cradle and drop = 4 cm


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 (μL)
5 80 80 0.5
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. Step one: This group obtained tray of sample tubes from the instructor (8 tubes containing a buffer 500 μL of buffer, 2 tubes with 1000 μL of SYBR green, 1 tube of water with 1000 μL at a pH of 8, and 5 tubes of calf thymus DNA labeled 0.25, 0.5, 1, 2 and 5.)
  2. Step two: Then obtained 8 tubes of patient DNA (3 tubes of patient 1 and 3 tubes of patient 2) and positive and negative controls.
  3. Step three: Pippette was then set to 80 μL and tip was attached before aspirating 80 μL of the 0.5 calf thymus DNA.
  4. Step four: The DNA was then ejected onto the dot in the middle of the super hydrophobic portion of the slide and the tip was ejected from the pipette.
  5. Step five: The light of the fluorimeter was then turned on and aimed at the sample.
  6. Step six: A camera was then focused on the sample, and with the box completely closed around the fluorimeter, used to image the droplet which formed on the slide from a side angle.
  7. Step seven: The group then repeated steps three through six for all samples including the DNA and control samples.


Data Analysis

Representative Images of Negative and Positive Samples

Name: Negative Control

Negative Control Image

Name: Positive Control

Positive Control Image


Image J Values for All Calibrator Samples


Name:table 1

Name:table 1
Calibration curve

DNA SYBR .PNG

PCR Results Summary

  • Our positive control PCR result was 13.647 μg/mL
  • Our negative control PCR result was 8.952 μg/mL

Observed results

  • Patient #52130 (AKA: 1A, 1B, and 1C):
    • Qualitative: Through fluorescence, it displayed a color of a light turquoise hue when exposed to the flourimeter's light in the black box and is clear throughout the droplet with no fogginess adn is displaying reflective lighting in the

water. In the gray scale, it is represented as a light gray droplet where image didn't change in display.

    • Quantitative: the final concentration values for 1A, B, and C are as follows: -3.177163938, -1.950868687, 2.939706515. The negative values are due to an error that most likely originated in poor image quality and, from there, was propagated throughout the analysis. There is also error in the negative control concentration because its value is larger than that of the patients. Based on lab theory - negative concentration is less than positive concentration - patient #52130 has concentrations that are nearer to the negative value.
  • Patient #17921 (AKA: 2A, 2B, and 2C):
    • Qualitative: Through the lights of the fluorescence, the droplet had a color of deep royal blue as the light of of the fluorimeter was passing though the liquid and only lighted the droplet's edges and exterior arcs outlining the it was physically present but left a black dark center on the drop unexposed to light. In the gay scale, the scale displayed the droplet as barely there except for a dot of light and the droplet is close to contextualized as a black color.
    • Quantitative: the final concentration values for 2A, B, and C are as follows: 2.8478477, 5.832963718, 4.228469769. Based on the logic used to determine patient #52130 negative, patient #17921 has values that also indicate that they are negative.



Conclusions

  • Patient 52130 (Patient 1):

Patient 1 DNA was split up into three samples of varying concentrations where they were close to the negative control concentration. When the three droplets concluded in concentrations way below the positive control meaning that there was no diseased primer present in the droplet even with the mixture of 80 μL of SYBR Green 1. With the fluorescence test where it lit up as a dark blue as the fluorimeter's light was refracted among the primers resulting in perfect matches of the divided DNA during PCR. This mixture of DNA and SYBR Green undergoes PCR where human DNA is denatured and a healthy primer then binds to it during annealing process since the base nucleotides match each other and anneal together as base pairs. With this visual presentation is accurate to the quantitative data calculated in which patient #52130 has a nice amount of DNA by 80μL and that it didn't glow the green dye from SYBR which is an indicator of a diseased primer duplicated during PCR.Patient 52130 resulted in not having a diseased primer

  • Patient 17921 (Patient 2):

Patient 2 DNA was split up into three samples of varying concentrations where they were close to the negative control concentration. When the three droplets concluded in concentrations way below the positive control meaning that there was no diseased primer present in the droplet even with the mixture of 80 μL of SYBR Green 1. With respect to the same occurring result from Patient 1, the fluorescence test where it lit up as a dark blue as the fluorimeter's light was refracted among the primers resulting in perfect matches of the divided DNA during PCR. This mixture of DNA and SYBR Green undergoes PCR where human DNA is denatured and a healthy primer then binds to it during annealing process since the base nucleotides match each other and anneal together as base pairs. With this visual presentation is accurate to the quantitative data calculated in which patient #52130 has a nice amount of DNA by 80μL and that it didn't glow the green dye from SYBR which is an indicator of a diseased primer duplicated during PCR. Patient 17921 does not carry a diseased primer at all.




SNP Information & Primer Design

Background: About the Disease SNP

First there is SNP which stands for "Single nucleotide polymorphism". A nucleotide is a molecule or molecules which link together to form the building blocks of DNA & RNA. Then Polymorphism is the presence of 2 or more distinct phenotypes in a population due to the expression of different alleles of a given gene, as human blood groups O, A, B & AB.

In SNP with a generic variation of rs 268, found in Homo Sapiens and has a chromosome variation mapped on Chromosome:8: 19956018. The clinical significance of this particular SNP is that it's Pathogenic and is associated as Lipoprotein Lipase or LPL and is also linked to SNP as a CHD disease.

The disease CHD also known as coronary heart disease is a common heart disease in which is the number one cause of deaths in the United States. The disease starts with having a waxy substance known as plaque building up in the coronary arteries. Nevertheless, Lipoprotien Lipase is a specific sequence of SNP that has specific functions like apolipoprotein binding, heparin binding and lipoprotein lipase activity.

An allele is a one of a # of alternative forms of the same gene through mutation on the chromosome that carries the genetic information passed on from parents. The disease-associated allele is classified with nucleotides as AGT. Within SNP, it is located in the numerical position as number 19956018 and then the reverse primer is located at the numerical position of 19956218.

Primer Design and Testing

The primer test was successful in receiving clear matches for both the forward and reverse primers when they would go through the PCR experiment and is in respect to working in a solution of 50 mM salt and 50 nM annealing oligo concentration. In the human genome there would be no matches with the diseased primer having a mutation of AGT which doesn't exist in the DNA. Since the human genome is unique with all of its characteristics, it is also at risk of obtaining coronary heart disease with a slight variation in the alleles or nucleotide build-up. The non-diseased primer would appear with results because there is no variations of the DNA and will have the AAT nucleotide instead and ended up in passing the primer test with matches.

Non-diseased Primer:
PCR gene.PNG Primer Temps.PNG

Diseased Primer:
Disease .PNG