BME100 f2013:W1200 Group6 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 Jenny Chen Role: Open PCR Machine Engineer Tracy Lopez Role: Protocol Planner Nayobe Bivins Role: Research & Development Specialist Alex Bugarin Role: Open PCR Machine Engineer Nicholas Kilpatrick Role: Protocol Planner LAB 1 WRITE-UP Initial Machine Testing The Original Design A diagram of the OpenPCR machine with each part labeled. The OpenPCR machine is a self-built machine capable of accurately maintaining and measuring temperatures for polymerase chain reactions. It comes with an application for the computer that helps in designing protocol and displays a user friendly interface showing the temperature of the thermocycler. There are several key parts that allow this machine to work in an orderly fashion. This includes the heating lid, the heating block, the LCD Screen, the heater, the circuit board, and the fan. The PCR Machine heats up the heating block that contains the test tubes with the samples of DNA with the heater, while the heating lid prevents the heat from escaping. During the denaturing and annealing of the DNA, the DNA continues to replicate causing a large amplification of the the certain strand of DNA. This process is used for gene amplification and can be used to create many copies of the same DNA. This can be used to see if there are infections present or for finding gene variants that can show whether or not a patient is easily susceptible to certain diseases. Experimenting With the Connections Unplugging the wires from the circuit board caused the display screen on the machine to turn off, resulting in the PCR Machine not being able to function and making the data unreadable. Unplugging the white wire that connects the circuit board to the heating block caused the temperature on the screen to decrease from 60°C to -40°C. This wire must be the temperature regulator and without this essential part the machine can not read temperatures correctly. Test Run This machine was first tested 10/23/13. As the machine went through the cycles, the temperature changed according to the numbers that were inputed into the computer. The temperature on the computer screen was the same as the one on the PCR Machine throughout all the cycles. Overall the machine ran smoothly and completed the 25 test run cycles giving it a passing mark. Protocols Thermal Cycler Program DNA Sample Set-up Positive control "PC" Patient 1 ID: 47569 "1" Patient 1 ID: 47569 "2" Patient 1 ID: 47569 "3" Negative control "NC" Patient 2 ID: 67318 "A" Patient 2 ID: 67318 "B" Patient 2 ID: 67318 "C" We will be testing 2 patients using PCR. Patient #1 is 47569 and patient #2 is 67318. Test tubes for patient 1 will be labeled: 1, 2 and 3. Test tubes for patient 2 will be labeled: A,B and C. Each patient will have 3 test tubes. There will also be a positive control and a negative control. These test tubes are labeled PC and NC respectively. DNA Sample Set-up Procedure Step 1: Label all PCR micro tubules according to above designated labels Step 2: Micro-pipette 50 µL of PCR reaction mix into each tubule Step 3: Micro-pipette 50 µL of designated DNA primer mix to designated tubules We will use the following heating and cooling protocol: The lid will be heated to 100°C. We will use an initial step at 95°C for 3 minutes. We will run 35 cycles to denature, anneal and extend. It will denature at 95°C for 30 seconds. It will anneal at 57°C for 30 seconds. It will extend at 72°C for 30 seconds. There is a final step at 72°C for 3 minutes. The final hold will occur at 4°C. PCR Reaction Mix 50 µL of each; Taq DNA Polymerase, MgCl2 and dNTP's DNA/ primer mix 50 µL of different template DNA in 8 tubes all eight test tubes will have the same forward and reverse primers Research and Development Working on it..... PCR - The Underlying Technology PCR Components and their Function The PCR (polymerase chain reaction) technique requires the following main components: template DNA, primers, Taq polymerase, magnesium chloride (MgCl2), and deoxyribonucleotides (dNTP’s). The DNA template contains the target DNA sequence that a researcher wants to amplify; the DNA sequence can be from an individual, animal, plant, or microorganism. Primers are tiny segments of DNA that bind to a specific site (i.e. on either end of the single-stranded DNA) of the target DNA sequence to initiate the replication of the target DNA sequence. Two primers are usually used in a PCR experiment so that one primer will attach on the top of the DNA strand while the other primer will attach to the other end. When primers are done binding to the DNA strand, a specific type of enzyme called Taq Polymerase is activated. This enzyme helps to helps synthesize new strands of DNA that are identical to the target DNA sequence. A buffer called magnesium chloride (MgCl2) is added to the PCR tube to stabilize the DNA strand. Lastly, deoxyribonucleotides such as adenine (A), thymine (T), cytosine (C), and guanine (G) are basically the building blocks for creating new strands of DNA. Steps of Thermal Cycling (Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the Q&A's from Section three of your worksheet) (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.)