BME103 s2013:T900 Group5

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Alexander Oropel: Research and Development Scientist Name: Matt McClintock Experimental Protocol Planner Name: student Role(s) Name: student Role(s) Name: student Role(s) 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 ... (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 The program ran, from a PCR application for Windows computer was as follows: Stage One: 1 Cycle, 95 °C for 180 seconds Stage Two: 35 Cycles, 95 °C for 30 seconds, 57 °C for 30 seconds, 72 °C for 30 seconds Stage Three: 1 Cycle, 72 °C for 180 seconds Final Hold: 4 °C DNA Sample Set-up The DNA was taken from two patients and separated into three replicates. This setup is accurate to how the vials were oriented in the PCR, and are labeled as such: +: Positive Control -: Negative Control 1:Patient 1 Replicate 1 2:Patient 1 Replicate 2 3:Patient 1 Replicate 3 4: Patient 2 Replicate 1 5: Patient 2 Replicate 2 6: Patient 2 Replicate 3 + - 1 2 3 4 5 6 DNA Sample Set-up Procedure 1.) Use the micro-pipet to dispense 50 μL of PCR reaction mix into 8 vials, be sure to change the transfer pipettes to avoid cross contamination. 2.) The DNA is transferred to its appropriate vial, 50 μL each. 3.) The vial labeled (+) is then given cancer DNA as a positive control for the cancer marker testing and the vial labeled (-) is given no DNA template, then the DNA is dispensed according to the above table. 4.) The vials are ready for the PCR system, which should be programmed according to the Cycler program above. PCR Reaction Mix The PCR reaction mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer and reverse primer. The PCR reaction mix contains necessary reactants for amplification to occur. It also makes it easy to transfer the DNA after amplification. DNA/ primer mix The primer is a determinant of the overall outcome of the PCR mix. The primer has distinct characteristics such as: -18-24 Bases Long - Balanced distribution of G/C and A/T bases - Not complementary to each other (5'-3') This primer acts to bind to the DNA Research and Development Specific Cancer Marker Detection - The Underlying Technology Polymerase Chain Reaction, or PCR, is the occurrence in which a segment of DNA is replicated multiple times. For the reaction that we are trying to produce the solution we will be using will contain Template DNA, Primers, Taq Polymerase, Magnesium Chloride and dNTP's. To begin the process of PCR the solution containing template DNA we take the template DNA and heat up the solution to 95°C for 30 seconds in order to break the hydrogen bonds between the nucleotides. This exposes the bases to allow the primers, which are short sequences of DNA that have been artificially synthesized, to attach to the exposed bases on the template DNA. In this process there are forward primers and reverse primers that attach to the exposed template DNA. After the primers are added, the solution is cooled down to 57°C in order to initiate primer annealing, or binding, to their respective regions. To complete a full strand of DNA the solution must be raised up to 72°C. The raise in temperature activates the Taq polymerase. Taq Polymerase is a protein that allows for binding of dNTP which are individual bases floating around within the solution that act as the subunits that the polymerase use to build new strands. Within the solution the dNTP's bind to the exposed bases starting from the primers. To aid in this process, the added Magnesium Chloride acts as a cofactor to allow polymerase to work and aids with the binding process. Taq polymerase will seek out and react with double stranded parts of DNA and can bind after it attracts individual bases which attach to the primer through a covalent bond, thus creating a new strand of DNA. In a PCR reaction this entire process is repeated multiple times, preferably 30 in order to cancel out other genes and work just for genes that code for cancer. The reason that the process only codes for the cancerous genes is that the primers were artificially synthesized in order to code for those genes. For the non-cancer gene there will be no binding of the primer since it does not match the same sequence of DNA as the cancer gene. As a result the non-cancerous gene will remain uncoded while the cancerous gene is continuously copied which allows for the development of an new strand by the Taq polymerase and the dNTP's within the solution. (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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)