BME103:T930 Group 1 l2
(→Thermal Cycler Engineering)
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Revision as of 19:40, 28 November 2012
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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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.
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.
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
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.
Research and Development
Background on Disease Markers
"GoTaqÂ® Colorless Master Mix (M714) Product Information." GoTaqÂ® 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>.