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 and Development Specialist Alex Bugarin Role: Open PCR Machine Engineer Nicholas Kilpatrick Role: Protocol Planner LAB 1 WRITE-UP Initial Machine Testing The Original Design The OpenPCR machine is a self-built machine capable of accurately maintaining and measuring tempratures 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 several key parts that contribute in having this machine working in orderly fashion. There is the heating lid, heating block, LCD Screen, Heater, Circuit Board, and Fan. Experimenting With the Connections When we unplugged the wires from the circuit board, the display screen on the machine turned off. Resulting in the PCR Machine not being able to function or turn on. When we unplugged the white wire that connects from the circuit board to the heating block, the temperature on the screen decreased 60°C to -40°C. This wire must be the temperature regualtor and without this essential part he machine can not read temperatures correctly. Test Run First tested 10/23/13. As the machine went through the cycles, the temperature was changing according to the numbers that was inputed into the computer. Also the temperature on computer screen was the same on the PCR Machine thoughout all the cycles. Overall the machine ran smoothly and completed the 25 test run cycles resulting in it being marked as passed. 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 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.)