OUR TEAM
 Name: Wade Patrick Machine Engineer |  Name: Liann Klein Machine Engineer |  Name: Haylee Poncy Protocol Planner |  Name: Kyle Labban Protocol Planner |  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
Key Features
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
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 the proper solution.
3. Using the fluorimeter equiptment, add two drops of each sample, followed by two drops of SYBR green.
4. Close the system down, preventing any light from entering the system, and record a photo to visually measure the presence of a positive or negative result.
5. Using a different pipette, clear the sample from the glass tray, move the tray forward, and repeat with the next sample.
6. Continue until all samples have been measured and photographed.
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
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.
Illustration
|
BME 103 Fall 2012
