BME103:T130 Group 5 l2

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(Thermal Cycler Engineering)
(Thermal Cycler Engineering)
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'''System Design'''<br> [[Image:BME103_group5_wells.jpg|350px]]  
'''System Design'''<br> [[Image:BME103_group5_wells.jpg|350px]]  
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<br> 8 wells were added, elongating the length of the lid <br>
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<br> 8 wells were added<br>
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[[Image:openpcrlid.png|350px]]<br>
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The additional wells will also elongate the lid
'''Key Features'''<br> The major change of the PCR machine was that eight extra wells were added to increase the sample size from 16 to 24. With the addition of the extra wells, the machine will be able to run more samples.  
'''Key Features'''<br> The major change of the PCR machine was that eight extra wells were added to increase the sample size from 16 to 24. With the addition of the extra wells, the machine will be able to run more samples.  

Revision as of 20:30, 28 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Name: Wade PatrickMachine Engineer
Name: Wade Patrick
Machine Engineer
Name: Liann KleinMachine Engineer
Name: Liann Klein
Machine Engineer
Name: Haylee PoncyProtocol Planner
Name: Haylee Poncy
Protocol Planner
Name: Kyle LabbanProtocol Planner
Name: Kyle Labban
Protocol Planner
Name: Alexandria LamR&D Scientist
Name: Alexandria Lam
R&D Scientist

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.


System Design

8 wells were added


The additional wells will also elongate the lid

Key Features
The major change of the PCR machine was that eight extra wells were added to increase the sample size from 16 to 24. With the addition of the extra wells, the machine will be able to run more samples.


Instructions





Protocols

Materials

Supplied in the Kit Amount
PCR Machine 1
Extra screws 5
CD containing programming application 1
Operations instruction manual 1
10 ft Extension cord 1


Supplied by the User Amount
Standard sized test tubes 16
DNA Primer Amounts vary per experiment
DNA Samples Amounts vary per experiment
Computer 1
Pipettes 16
Sybr Green Amounts vary per experiment
Refrigerator 1
Power source N/A

PCR Protocol

Kyle - Create a step-by-step procedure for setting up and running PCR reactions. Your instructions should include everything from adding reagents to the tubes, to programming the PCR machine and running the reaction.


DNA Measurement Protocol

  1. Collect samples generated from "PCR Protocol".
  2. Using separate pipettes for each individual sample, transfer the 150μL into the larger test tubes containing 400 mL of the buffer solution.
  3. Set up the fluorimeter machinery as instructed, ensuring that the system is devoid of any light, as it may prevent accurate readings. Use a "blank" sample using distilled water to ensure all machinery and processes are in order.
  4. Using the fluorimeter equipment, add two drops of each sample onto the glass plate, followed by two drops of SYBR green. When placing the drops, one should ensure that they are initially spaced out, as they will combine when more substrate is added.
  5. Close the system down, again preventing any light from entering the system. To record the results, a photo will be used to visually measure the presence of a positive or negative result. The fluorimeter set comes with a stand to enable a SmartPhone to be utilized. Individuals will obtain the most accurate results by setting the ISO at 800 and turning off the flash setting.
  6. Using a different pipette for waste products, clear the sample from the glass tray, move the tray forward, and repeat with the next sample. The waste samples can be placed in a separate plastic cup, and eventually disposed of in a biohazard bin.
  7. Repeat this process until all samples have been measured and photographed.

Fluorimeter Set Up
Fluorimeter Set Up

Photo origin: http://openwetware.org/wiki/BME103:T130_Group_9


Positive Result
Positive Result
Negative Result
Negative Result

Research and Development

Bayesian statistics

A=Alzheimer's B=Positive test Result

P(A / B) = P(B / A)P(A) / P(B)

Background on Disease Markers

Alzheimer's disease is a form of dementia that occurs with loss of brain function. It affects multiple areas of the brain associated with memory, language, personality, perception, and cognitive skills. The disease typically manifests itself through forgetfulness, but gradually progresses to inability to perform basic functions, speak, and recognize family members. Currently, there is no cure. Treatment tries to slow down the disease or at the least, manage symptoms.

An SNP related to Alzheimer's disease is rs1466662 (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1466662). It is located on chromosome four, the intron region of NM_001142552.1 and arises from a missense mutation replacing an A with a T. It is the most significant SNP outside of the SNP linked to APOE.


Primer Design

The backwards primer is TAT TTT TAG AAG CGA TAA AA. The forwards primer is GCC TCT TTG CCC TCT GTT TT. An allele not containing the disease will not have the sequence that allows the primers to bind. If the primers cannot bind, then that means Taq polymerase does not know where to bind. If Taq polymerase does not bind, then the sequence does not get replicated. Therefore, there will be no PCR product. Conversely, if the disease allele is present, the primers will bind. Taq polymerase will then be able to bind to the DNA and replicate the strands, creating more double-stranded DNA yielding a PCR product.


Illustration


Image:pcrcopies.gif

The wanted gene in the figure above refers to rs1466662.

Photo courtesy of http://users.ugent.be/~avierstr/principles/pcr.html


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