BME100 f2013:W900 Group9 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: Julianna A. Brunner Role: Open PCR Machine Testing Name: Neel J. Patel Role: Open PCR Machine Testing Name: Callie M. LaMarche Role: Protocol Planning Name: Allison Lind Role: Research and Development Name: Sydney M. Lankford Role: Protocol Planning LAB 1 WRITE-UP Initial Machine Testing The Original Design The Open PCR is a machine that uses heating and cooling to amplify DNA strands. The DNA heats to 95 degrees Celsius, seperating the double helix structure of the DNA. Then, it cools down to 57 degrees Celsius to attatch the primers to the begining of the desired strand. The final step of the cycle reheats the DNA to 72 degrees Celsius. This attaches the DNA polymerase, an enzyme which attatches the complimetary bases to the template DNA strand, to the primer, replicating the DNA. After 35 cycles of replicating, there is an abundance of the desired strand of DNA, outnumbering the initial DNA. Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine screen did not turn on. (This was the connection between the screen and the circuit board.) When we unplugged the white wire that connects (part 6) to (part 2), the temperature sensor did not work. It read -40 degrees Celsius rather than 24 degrees Celsius. Test Run The Open PCR machine was tested for the first time on October, 23, 2013 at approximately 10:15 a.m. The circuitry of the machine is fairly complex however the various parts of the machine were defined and comprehended before the experimention began. The Open PCR machine ran at an average rate, about two hours a test or three and a half minutes a cycle, with accurate cycling temperatures. Protocols Thermal Cycler Program Initial denaturation step: one cycle at 95°C for 3 minutes. Denaturation step: 35 cycles at 95°C for 30 seconds. Anneal step: one cycle at 57°C for 30 seconds. Extended step: one cycle at 72°C for 30 seconds. Final extension step: one cycle at 72°C for 3 minutes. Final holding: refrigeration of DNA at 4°C. DNA Sample Set-up Positive Control: cancer DNA template Tube label: PC Patient 1 ID: 46398 Replicate 1 Tube label: P11 Patient 1 ID: 46398 Replicate 2 Tube label: P12 Patient 1 ID: 46398 Replicate 3 Tube label: P13 Negative Control: non-cancer DNA template Tube label: NC Patient 2 ID: 73945 Replicate 1 Tube label: P21 Patient 2 ID: 73945 Replicate 2 Tube label: P22 Patient 2 ID: 73945 Replicate 3 Tube label: P23 DNA Sample Set-up Procedure Step 1 : Gather supplies and label test tubes (as shown above with the table). Step 2 : Add Primer 1 to each PCR test tube, which attaches to either end of the DNA at hand. Step 3 : Add Primer 2 to each PCR test tube, attaching to the second site of the DNA strand. Step 4 : Add Nucleotides to each PCR test tube. Step 5 : Add DNA Polymerase to each test tube. Step 6 : Put PCR test tubes into thermocycler. Collect data. Step 7 : Store test tubes for later use. PCR Reaction Mix 8 PCR reaction mix tubes with 50 μL each. Each tube contains Taq DNA polymerase, MgCl2, and dNTP's. DNA/ primer mix 8 DNA/ primer mix tubes with 50 μL each. Each contains a different template DNA. All tubes will have the same forward primer and reverse primer. Research and Development PCR - The Underlying Technology Polymerase Chain Reaction (or PCR) is a procedure used to create copies of DNA. DNA BASE-PAIRING Deoxyribonucleic acid (DNA) COMPONENTS PCR is composed of multiple steps, each essential to the overall process. (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.) Sources: http://openpcr.org/what-is-pcr http://www.ehow.com/about_5390976_magnesium-chloride-used-pcr.html