BME100 f2013:W1200 Group1 L4

BME 100 Fall 2013	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Chloe Kwon Role: Research and Development Name: Nima Afzalian Role: Open PCR Machine Testing Name: Casey Weinstein Role: Open PCR Machine Testing Name: Jeff Miles Role: Protocol Planning LAB 1 WRITE-UP Initial Machine Testing The Original Design The image shown above is of a device called an OpenPCR machine. An OpenPCR machine is a low cost, yet highly accurate thermocycler capable of performing polyermase chain reactions that amplifies a specific target DNA segment while maintaining extreme differences in temperature change. After going through many cycles of heating and cooling, the OpenPCR machine replicates a single strand of DNA into many copies. As seen in the image, the OpenPCR machine is connected to a computer using a USB cable. The computer uses an OpenPCR program that allows the number of cycles and each step and desired temperature and time to be adjusted accordingly. Thus, the OpenPCR machine begins the process according to the information inserted into the program. The image shown above is of the inside of the OpenPCR machine. The environmental changes of heating and cooling are controlled by the heater, heating lid, and cooling fan. The data collected is computed and transferred from the circuit board to the LED screen on top of the OpenPCR machine. Experimenting With the Connections The PCR machine is heavily dependent on numerous connections and and interconnected parts. This means that, in order for it to work well, everything must be in its proper location and plugged in. This being said, when we unplugged part 3 (the LCD screen) from part 6 (circuit board) of the machine, the connection from the LCD to the circuit board was severed, resulting in the screen turning off. Also, when we unplugged the white wire that connects part 6 (circuit board) to part 2 (core) of the machine, the temperature changed from 26.7 degrees Celsius to -40 degrees Celsius. It is very important that all connections are correct in order to have an effective PCR machine. Test Run Our group's PCR machine was tested on October 23. The test began at 1:08 pm. After 30 minutes, 8 cycles were completed. Protocols Thermal Cycler Program Initial Step 95°C for 3 minutes: The initial Template DNA strand separates. Denature 95°C for 30 seconds: The DNA double helix separates, creating two single-stranded DNA molecules. Anneal 57°C for 30 seconds: The two primers attach to opposite ends of the top and bottom strand of the target DNA segment. Extend 72°C for 30 seconds: The DNA polymerase activates. Replication of the target DNA segment begins. Final Step 72°C for 3 minutes: The complementary binding of nucleotides continues until it gets o the end of the DNA strand and falls off. Final Hold 4°C: The PCR reaction ends. DNA Sample Set-up Tube A: Positive Control: cancer DNA template Tube B: Patient 1 ID-16946 Replicate 1 Tube C: Patient 1 ID-16946 Replicate 2 Tube D: Patient 1 ID-16946 Replicate 3 Tube E: Negative Control: non-cancer DNA template Tube F: Patient 2 ID-46296 Replicate 1 Tube G: Patient 2 ID-46296 Replicate 2 Tube G: Patient 2 ID-46296 Replicate 3 DNA Sample Set-up Procedure Step 1: Fill each reaction tube with 50μL of the PCR reaction mix and label all tubes according to the chart above Step 2: Add 50μL of DNA sample mix to the corresponding reaction tube (Note: use each disposable pipette tip only once to avoid cross-contamination) Step 3: Place to reaction tubes into the thermocycler and run the above program PCR Reaction Mix The PCR reaction mix will contain 8 tubes. Each tube will contain 50μL of the following: Taq DNA polymerase MgCl2 dNTP's DNA/ primer mix The DNA primer mix contains 50μL of the following: an extracted sample of the patient's DNA forward primer reverse primer Research and Development PCR - The Underlying Technology (Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) What is the function of each component of a PCR reaction? Template DNA is the sample DNA that contains the target DNA segment. Two primers are designed to match to the segment of desired DNA to copy. One primer attaches to the top strand at one end of the target DNA segment and the other primer attaches to the bottom strand at the opposite end. Taq Polymerase is an enzyme that synthesizes new strands of DNA complimentary to the target strand. It can generate new strands of DNA using a DNA template and primers. It is heat resistant. Magnesium Chloride is how magnesium is added to the PCR reaction. It acts like a catalyst. It is not consumed in the reaction but is a necessary component. The more Magnesium Chloride is added, the faster the reaction goes. Deoxyribonucleotides provide the energy and nucleotides for the synthesis of DNA. The nucleotides include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). They are collected by the Taq Polymerase and used in complementary sequencing of the template DNA strand. What happens during each step of thermal cycling? The initial step of thermal cycling takes place at 95°C for 3 minutes. In this step, the single-stranded template DNA separates. (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)