BME103:W930 Group3

BME 103 Fall 2012	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: Ryan Bath Role(s) Open PCR Machine Engineer Name: Geon-Woo Kim Role(s) Experimental Protocol Planner Name: Troy Kozlowski Role(s) Open PCR Machine Engineer Name: Phillip Mercado Role(s) Experimental Protocol Planner Name: Eliza Normen Role(s) R&D Scientist Name: Jacob Swartz Role(s) R&D Scientist LAB 1 WRITE-UP (Please finish by 11/7/2012) Initial Machine Testing The Original Design write paragraph here Experimenting With the Connections If the Arduino UNO board is disconnected fron the LCD screen, then then LCD screen will not be able to display information; this includes information such as the current cycle of the PCR and the current tempereture. If the Arduino UNO board is disconnected from the 16-tube PCR block, then the LCD wouldn't be able to display any temperatures, this is because temperatures in the 16-tube block are not being monitored. Test Run The experiences of first testing the Open PCR machine, on October 24th, 1012, were of mixed results. As for the set up of the Open PCR, things went fairly well. Connecting the Open PCR to a computer was not a problem and neither was finding a suitable location to let the machine run. However, the first computer we connected the Open PCR to had problems running the Open PCR software. The experiment design part of the software would not allow for editing of the number of cycle, resting temperature, time length of the cycles and all other variables of the experiment. The software might have been corrupted or the computer may not have been running correctly; whatever the case, the Open PCR had to be moved to another computer in order to solve this problem. The use of the second computer allowed for editing of the experimental variables and the initiation of the experiment. However, further problems arose once the experiment was in progress. From the beginning of the experiment the Open PCR machine being used took a considerable amount of time on the cooling part of the cycle, much longer than the other groups running the experiment. If the machine does not cool down correctly and to the right temperature, then the PCR cannot move onto the next cycle. And eventually, due to this problem, the Open PCR machine became stuck on the cooling part of cycle 5 of 30 and would not move forward in the experiment. After trouble shooting from TA's and the professor the problem could not be reverse and additional amplified DNA samples will have to be created for group 3. Protocols Polymerase Chain Reaction Polymerase Chain Reaction is a biochemical technology that is used in molecular biology to amplify single/multiple copies of a piece DNA, generating thousands to millions of copies of a targeted DNA sequence. To do so, PCR relies on thermal cycling, which consists of repeated cycles of heating and cooling the samples in order to melt the DNA and have the enzymes replicate the targeted strand if found. Primers, which are short DNA fragments, have complementary sequences to the target strand of DNA, in addition to a DNA polymerase, which allows selective and repeated amplification of the target strand. As the cycles progress, the DNA is used as a template for exponential amplification (or creation of copies). Steps of how to amplify a patient's DNA sample Step 1: Initialization The initialization step consists of heating the reaction to a temperature between 94 to 98°C, depending on how thermostable the polymerase is. It is held at this temperature for 1-9 minutes. Note, however, that this step is only required for DNA polymerases that require a "hot start", which reduces non-specific amplification during the set-up stage of the PCR. Step 2: Denaturation The detanuration step is the first cycle which consists of heating the reaction to 94-98°C for 20-30 seconds. This causes the DNA template to melt by disruption of the hydrogen bonds between complementary bases, which yields single-stranded DNA. Step 3: Annealing The annealing step consists of lowering the temperature to 50 to 65°C for 20 to 40 seconds, which allows annealing of the primers to the single-stranded DNA template. Annealing is the process of heating then cooling the DNA strands so as to separate the double-strands into single-strands. Normally, the annealing temperature is about 3 to 5°C below the melting temperature of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. Step 4: Initial Extension Initial extension Step 5: Final Extension Final extension Step 6:Final Hold Final hold Reagent Volume Temple DNA (20 ng) 0.2 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0 μL GoTaq master mix 50.0 μL dH2O 47.8 μL Total Volume 100.0 μL Flourimeter Measurements Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (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.) Results (Your group will add the results of your Fluorimeter measurements from Week 4 here)