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 Eight wells were added, this is a good amount to increase sample size significantly yet not too drastic of a change that will affect the size by making it too large. The additional wells will also slightly 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. This also affects the size as the additional wells will cause it to be longer than the original design. Instructions The instructions for assembling the PCR machine will mostly stay the same the only difference will be that the screws will be located in a slightly different place than the original design. 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 Using a pipette, transfer 1.0-1.5 micro liters of the DNA sample into the desired number of test tubes. Pipette approximately 3 micro liters of reagent solution into each of the test tubes with the DNA. Invert tubes, then turn upright to mix solution. Plug in OpenPCR Machine and turn it on. Open lid and place tubes into holder in PCR machine (the machine can now hold a maximum of 24 test tubes). Close the lid. Add the following cycles on the OpenPCR program: Stage 1: 1 cycle, 95 degrees Celsius for 3 minutes Stage 2: 30 cycles, 95 degrees for 30 seconds, 57 degrees for 30 seconds, 72 degrees for 30 second Stage 3: 72 degrees for 3 minutes Hold: 4 degrees Run reaction. After the program is complete, open the lid and remove samples for further analysis. DNA Measurement Protocol Collect samples generated from "PCR Protocol". Using separate pipettes for each individual sample, transfer the 150μL into the larger test tubes containing 400 mL of the buffer solution. 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. 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. 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. 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. Repeat this process until all samples have been measured and photographed. Fluorimeter Set Up Photo origin: http://openwetware.org/wiki/BME103:T130_Group_9 Positive Result Negative Result 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 The wanted gene in the figure above refers to rs1466662. Photo courtesy of http://users.ugent.be/~avierstr/principles/pcr.html