BME100 f2014:Group8 L5

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OUR TEAM

Robert Schultz
Isabella Germinario
Bakir Mousa
Daniel Hersey
Ethan Lloyd
Bailey Myers

LAB B/C

PROCEDURE

Camera Settings:

  • Phone type: Iphone 4
  • Flash off
  • ISO Setting:Auto
  • White Balance:Auto
  • Exposure:Auto
  • Saturation:Auto
  • Contrast:Auto

Calibration: -The camera was placed 4 cm from the fluorimeter. The camera was aligned so that it was level with the fluorimeter. After this, a blackout box was placed over the camera and sample so that there was darkness when the photo was taken.

Solutions Used For Calibration

Concentration chart bme100 lab 5.PNG

Part B

To begin, each group was provided with three DNA samples, from two different patients. First, a positive control tube of DNA was produced by micropipetting 50 microliters of PCR reaction mix into an empty tube, then using a clean tip to micropipette the positive control DNA sample into the same tube. This yielded 100 microliters of solution. The same procedure was followed for the negative control DNA, and replicates 1, 2, and 3 for both patients 1 and 2. Then the tubes, each full of 100 microliters of PCR solution, were placed in the PCR heating block. The PCR cycles ran while the group cleaned up the equipment that was used, and the finished data would be acquired in Part C.

Part C


First, the camera of an iPhone 5C was calibrated in relation to the correct white balance and contrast necessary to capture the images of the solution drops. The camera was placed inside the cradle, and the slide height was adjusted to be at a right angle with the camera, and at the correct height for the camera to capture images of the drop from the side. The distance between the smartphone and cradle was 5 centimeters.

Next, 80 microliters of SYBR Green I solution was micropipetted onto the slide in the middle of the first two rows. Then 80 microliters of the calf thymus or water blank solutions were added to the drop. Once the drop was pinned on the slide, the slide was adjusted so that the blue LED light was focused by the drop onto the fiber optic fitting on the other side of the drop. The timer on the group's camera could not take a picture without creating a flash, so one team member had to take pictures with their hand inside the light box, preventing as much light as possible from entering and interfering with the image. Three images of each drop were taken. The pipettor was used to remove all 160 microliters of solution from the slide, and the procedure was repeated for each concentration of calf thymus DNA given by the instructions.

In continuation of the PCR reactions which were set up in part B, the PCR samples were obtained and measured using the fluorimeter. The micropipettor was set to 120 microliters and all 100 microliters of PCR sample were transferred into the tubes which contained buffer solution. This procedure was followed for the rest of the PCR sample tubes as well. Next, 80 microliters of the PCR solution which had been diluted by the buffer solution was pipetted onto a slide, along with 80 microliters of SYBR GREEN I, and again, three images of each drop were captured. All tubes were disposed of in hazardous waste bags, and equipment was returned after fluorimeter analysis was finished.


DATA ANALYSIS

Positive Control: Description of image

Negative Control: Description of image

Image J Values

Description of image Description of image

Calibration Curve Description of image

Data Summary:

  • From the data provided, it is likely that Patient 1 (ID# 95366) is positive for Cat Thymus DNA while patient 2 (ID# 41561) is negative.

LAB D

SNP INFORMATION AND PRIMER DESIGN

Part 1-

Vocabulary-

  • Nucleotides form the basic structural unit of nucleic acids such as DNA.
  • Polymorphism is the occurrence of a number of alternative forms within a section of a nucleic acid or protein molecule.

rs16991654-

  • What species is this variation found in? (Latin name)
  -The rs16991654 variation is found in Homo Sapiens.
  • What chromosome is the variation located on?
  -It’s located on the chromosome 21
  • What is listed as the Clinical significance of this SNP?
  -The clinical significance is pathogenic.
  • Which gene(s) is this SNP associated with?
  -It’s associated with the KCNE2 gene.
  • What disease is linked to this SNP?
  -The rs16991654 variation is linked to Long QT syndrome.

Part 2-

  • What does KCNE2 stand for?
  -Potassium  voltage-gated channel, Isk-related family, member 2
  • Briefly describe the molecular function of this gene.
  -This gene regulates the movement of potassium ions in and out of a system. It affects neuron function, heart function, pancreatic eyelet cells, and tissue covering.
  • What is an allele?
  -Set of nucleotides which affect the development of proteins and phenotypic variances in organisms.
  • The disease-associated allele contains what sequence?
  -CTC
  • The numerical position of the SNP is:
  -34370656

Part 3-

  • Non-disease forward primer (20 nt):
  - 5'-CATGGTGATGATTGGAATGT
  • The numerical position exactly 200 bases to the right of the disease SNP is:
  -34370856
  • Non-disease reverse primer (20 nt):
  - 5'-CCCTTATCGAGGGGGACATT
  • Disease forward primer (20 nt):
  - 5'-CATGGTGATGATTGGAATGC
  • Disease reverse primer (20 nt):
  - 5' -CCCTTATCGAGGGGGACATT