BME103:T930 Group 1 l2: Difference between revisions
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2. Download the PCR software onto the computer. <br> | 2. Download the PCR software onto the computer. <br> | ||
3. | 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. <br> | ||
4 | |||
. Prepare the following reaction mix on ice: | |||
{| {{table}} | |||
|- style="background:#f0f0f0;" | |||
| '''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 | |||
|- | |||
| dH<sub>2</sub>O || 47.8 µL | |||
|- | |||
| '''Total Volume''' || 100.0 µL | |||
|} <br> | |||
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. <br> | |||
6. Place the reactions in a thermal cycler that has been preheated to 95 degrees Celsius. Perform PCR. | |||
'''What Occurs In the PCR Machine''' <br> | |||
1. Denaturation: a 2-minute denaturation at 95 degrees celsius. <br> | |||
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. <br> | |||
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. <br> | |||
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. <br> | |||
5. Cycle Number: the optimal amplification is 25-30 cycles, but up to 40 may be performed. <br> | |||
Revision as of 14:56, 28 November 2012
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 TEAMLAB 2 WRITE-UPThermal Cycler EngineeringOur re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
Key Features
Instructions
ProtocolsMaterials
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 DevelopmentBackground on Disease Markers
Primer Design
Illustration
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