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 Name: Jenny Chen Role(s) Open PCR machine testers Name: Tracy Lopez Role(s) Protocol Planning Name: Nayobe Bivins Role(s) Research and Development Name: Alex Bugarin Role(s) Open PCR machine testers Name: Nicholas Kilpatrick Role(s) Protocal Planning LAB 1 WRITE-UP Initial Machine Testing The Original Design (Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is) Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer) Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) 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.)