BME103:W930 Group7

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: Jake Turner PCR Machine Engineer Name: Tyler Allen PCR Machine Engineer Name: Khalil Pathan Experimental Protocol Planner Name: Pahul Singh Experimental Protocol Planner Name: Frea Mehta Research and Development Specialist Name: Paul Song Research and Development Specialist <gflash>400 300 http://www.youtube.com/embed/x5yPkxCLads?rel=0</gflash> LAB 1 WRITE-UP Initial Machine Testing The Original Design This is a solidworks rendering of the OpenPCR machine. The OpenPCR is an affordable alternative to costly clinical machines used to amplify particular DNA sequences. This interfaces with any computer with the proper software downloaded and the process of thermal cycling to conduct a variety of tests. This could be anything from paternity tests to test for genetic marks of cancer. Experimenting With the Connections When we unplugged part (part 3) from (part 6), the machine did not have power. The blue display screen did not turn on and appeared completely black. When we unplugged the white wire that connects (part 6) to (part 2), the machine temperature on the display screen appeared incorrectly. Part 6 is responsible for recording the the internal temperature of the machine throughout the test. Test Run Our first Open PCR test was conducted on Wednesday, October 24, 2012. While running our open PCR test, we experienced nothing but problems. We set the cycles to the appropriate temperatures and time intervals; the Initial cycle on 95°C for 30 seconds, the Denaturing cycle on 95°C for 30 seconds, the Annealing cycle on 55°C for 30 seconds, the Extending cycle on 72°C for 30 seconds, the final cycle on 72°C for 180 seconds, and the final hold at 20°C. Initially, our open PCR appeared to be running correctly for the desired two hour time interval. However, due to a cycling error, our timer extended to nearly three hours. Not only did our test exceed the desired time interval, but our time would not wind down. Our test constantly moved up and down between the times of two hours thirty minutes and two hours and fifty minutes. When our time got close to two thirty, more time would be added to our test. Also, our laptop was experiencing errors. Our laptop received an application error notice multiple times, each time disrupting our process. As a result of these complications, when the two hours elapsed we only reached step seventeen of thirty. We were forced to prematurely end our test. Therefore, we could not receive sufficient results. Protocols Polymerase Chain Reaction ( 1. Within a polymerase chain reaction everything is controlled by temperature. The high temperature(95 C) causes melting of DNA templates and primers by disrupting the hydrogen bonds. Next is annealing. The temperature is dropped down to 65 temporarily(20 seconds) to allow a piece of DNA to bind to your product from the initial step. The polymerase binds to the DNA template and DNA synthesis begins. Next is elongation, the DNA polymerase synthesizes a new DNA strand. This process is repeated to replicate numerous strands of DNA. 2. 1. Heat denaturation- a. Heat the reactant , which causes melting of the DNA b. A DNA molecule sequence is targeted which is then separated into two strands c. Separation is because of hydrogen bonds breaking 2. Primer annealing a. Then you lower the temperature to 65 which allows a piece of the DNA to bind to the initial step product. b. Each strand of DNA molecule becomes annealed with an oligonucleotide primer complementary to either end of the target sequence. 3. Primers extension a. DNA polymerase is added and complementary strands are synthesized at 65-75 C b. Causes synthesis of a new strand in the direction of 5 to 3 direction 4. Reagent Volume Template DNA (20ng) .2 µL 10 µM forward primer 1.0 µL 10 µM reverse primer 1.0 µL GoTaq master mix 50.0 µL dH20 47.8 µL Total volume 100.0 µL ) Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology The NCBI database is used to isolate the sequence used and determine specific primers. (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 Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 267793 0 Negative PCR: Positive Control 27409968 2 Positive PCR: Patient 1 ID 91562, rep 1 3511064 0.238984 Negative PCR: Patient 1 ID 91562, rep 2 15099598 1.09290 Positive PCR: Patient 1 ID 91562, rep 3 8451848 0.603051 Negative PCR: Patient 2 ID 25235, rep 1 17311845 1.25591 Positive PCR: Patient 2 ID 25235, rep 2 9289657 0.200563 Negative PCR: Patient 2 ID 25235, rep 3 28825322 2.10429 Positive KEY Sample = Sample denotes the sample of DNA used in a given trial. Each sample represents one extraction of DNA from one of two patients. Integrated Density = DNA μg/mL = To calculate the concentration of DNA, we created a calibration curve using the given concentrations of DNA for the negative and positive control samples - 0micrograms/mL and 2micrograms/mL, respectively. This yielded the equation y = (X-267793)/(13571087.5) where y = the concentration of DNA in micrograms/mL and X = the integrated density for a given sample. Conclusion =