BME103:T130 Group 5 l2

BME 103 Fall 2012	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
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 The major change done here was that there was an addition of eight extra wells. The wells were added in order to increase the horizontal width of the plate. There are now 24 wells instead of the original 16. The additional wells allow more samples to be tested at a given time. 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 equipment, 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 [math]\displaystyle{ P(A/B)=P(B/A)P(A)/P(B) }[/math] 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:]]