BME100 f2013:W1200 Group18 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: Carlyn Harris Role(s):Protocol Name: Tori Platt Role(s):Reseach and Development Name: Nikhil Patel Role(s):Initial PCR Testing Name: Alexandra Olson Role(s): Protocol Name: Matthew Armas Role(s):Initial PCR Testing Name: student Role(s) 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's screen on top of the box no longer functioned. The screen shows the data of the experiment, but because it was unplugged, we could not see the data. When we unplugged the white wire that connects (part 6) to (part 2), the machine's heat sensor no longer functioned. It determines how hot the machine runs, therefore the machine did not pick up any heat when it was unplugged. Test Run Open PCR was first tested on 10/23/13 from 1:18pm to 1:44pm. During the lab, we inspected the 16-tube PCR block and the heating lid, which are both designed to retain a constant temperature across the samples that would be tested. We also explored the insides of the machine, and how its various components affected one another. Our experience with the machine was somewhat frustrating because the machine failed to complete the required amount of cycles in a reasonable amount of time. It was labeled as being slow. Protocols Thermal Cycler Program Lid heated to 100 degrees Celsius 1.Initial: Thermo-cycler reaches 95°C for 3 minutes. Here, the DNA double helix separates into 2 strands 2. 35 cycles of the following: -Denature at 95°C for 30 seconds. Double helix separates. -Anneal at 57°C for 30 seconds. Primers attach to their target strands -Extend at 72°C for 30 seconds.DNA polymerase is activated. It locates the primer and begins to add complementary nucleotides onto the strand until it gets to the end of the strand. At the end of these steps, you have numerous replicates of your target DNA 3. Final Step: 72°C for 3 minutes 4. Final hold: 4°C DNA Sample Set-up PCC= Positive control: Cancer DNA NCC= Negative control: Non-cancer DNA P11,P12,P13= Patient 1's DNA samples P21,P22,P23= Patient 2's DNA samples Patient 1 ID: 54174 Patient 2 ID: 43184 PCC P11 P12 P13 NCC P21 P22 P23 DNA Sample Set-up Procedure 1. Label 6 50 μL disposable pipette tips with the labels given below corresponding to each patient. 2. Label the remaining 2 50 μL pipette tips, one as the positive control cancer DNA (PCC) and one as the negative control non-cancer DNA (NCC). 3. Pipette the PCR reaction mix into each reaction tube. 4. Using a different disposable pipette tip for each sample, pipette the DNA sample primer mix into each reaction tube. 5. Place the reaction tubes in the thermal cycler. 6. Run the PCR software that follows the protocol below. PCR Reaction Mix The PCR reaction mix contains: -Tag DNA Polymerase -MgCl2 -dNTP's DNA/ primer mix The DNA primer mix contains: -Different template DNAs -Forward primer -Reverse primer Research and Development Polymerase Chain Reaction (PCR) - The Underlying Technology Polymerase chain reaction occurs when DNA is heated and cooled at exact temperatures for exact intervals of time. However, there are five components required for the reaction to work successfully; template DNA, primers, TAQ polymerase, Magnesium Chloride (MgCl2), and dNTPs (deoxyribonucletides). (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.)