BME103:T930 Group 1 l2

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 Nick Hool: Machine Engineer Joseph Heath: Machine Engineer, R&D Scientist Jessica Kemper: Protocol Planner Maile Ravenkamp: Protocol Planner Christian Boden: R&D Scientist LAB 2 WRITE-UP Thermal Cycler Engineering Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. System Design Key Features The first piece of this PCR machine that we changed was the power supply source. We decided to make it smaller and more compact. A smaller power source 1), makes the entire machine lighter and adds mobility and 2), greatly reduces the chance of the power source overheating and causing the machine to melt or catch fire. As a result of making the power source smaller, we had to change the design of one of the side panels. Instructions The construction of the modified open PCR machine isn't any different than the original steps to putting it together, there are just modified parts like a smaller more compact power supply that is more efficient and the body is made out of ABS plastic which can be rapidly prototyped using 3-D printers so that they technology of open PCR can become more readily available. Here is a link to a guide to putting together your open PCR machine. Media:PCR guide.pdf Protocols Materials Supplied in the Kit Amount PCR Machine 1 Positive Control DNA Sample 27282151 Negative Control DNA Sample 35526894 Patient 1 DNA Sample 19073943 Patient 2 DNA Sample 29391013 PCR: Patient 2 ID 11260 , rep 1 1903450 PCR: Patient 2 ID 11260, rep 2 5214727 PCR: Patient 2 ID 11260, rep 3 5099077 PCR Protocol 1. You will need a PCR machine and computer. 2. Download the PCR software onto the computer. 3.Thaw the GoTaq Colorless Master Mix at room temperature. Vortex the Master Mix, then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube. 4 . Prepare the following reaction mix on ice: Reagent Volume Template 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 5. If using a cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation during thermal cycling. Centrifuge the reaction mix in a microcentrifuge for 5 seconds. 6. Place the reactions in a thermal cycler that has been preheated to 95 degrees Celsius. Perform PCR. What Occurs In the PCR Machine 1. Denaturation: a 2-minute denaturation at 95 degrees celsius. 2. Annealing: perform the reaction about 5 degrees Celsius below the calculated melting temperature of the primers and increasing the temperature in increments of 1°C to the annealing temperature; this should occur anywhere between 30 seconds and 1 minute. 3. Extension: performed between 72-74 degrees Celsius, extension allows 1 minute for every 1 kb of DNA to be amplified; the suggested time for extension is 5 minutes. 4. Refrigeration: refrigerate the tubes at 4 degrees Celsius for several hours; this will minimize the opportunity for DNA polymerase to continue to be active at higher temperatures. 5. Cycle Number: the optimal amplification is 25-30 cycles, but up to 40 may be performed. DNA Measurement Protocol Research and Development Background on Disease Markers Primer Design Illustration References "GoTaq® Colorless Master Mix (M714) Product Information." GoTaq® Colorless Master Mix Protocol. Promega, 2012. Web. 15 Nov. 2012. <http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/>. Hunt, Margaret. "Real Time PCR Tutorial." Real Time PCR Tutorial. University of South Carolina, 10 July 2010. Web. 15 Nov. 2012. <http://pathmicro.med.sc.edu/pcr/realtime-home.htm>.