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Team:DTU-Denmark/Methods/PCR-touchdown
From 2013.igem.org
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# 98°C for 0:10 - Denaturing after each cycle | # 98°C for 0:10 - Denaturing after each cycle | ||
# 65°C for 0:30 - Annealing | # 65°C for 0:30 - Annealing | ||
| - | #* | + | #* Temperature increment of -0.5°C/cycle |
# 72°C for 2:00 - Elongation | # 72°C for 2:00 - Elongation | ||
| - | # Go to number 2 - repeat | + | # Go to number 2 - repeat 10 times |
# 98°C for 0:10 - Denaturing after each cycle | # 98°C for 0:10 - Denaturing after each cycle | ||
# 55°C for 0:30 - Annealing | # 55°C for 0:30 - Annealing | ||
# 72°C for 2:00 - Elongation | # 72°C for 2:00 - Elongation | ||
| - | # Go to number 7 - repeat | + | # Go to number 7 - repeat 30 times |
# 72°C for 5:00 - Final extension | # 72°C for 5:00 - Final extension | ||
# End | # End | ||
Revision as of 19:38, 26 June 2013
How to do "touchdown-PCR"
Touchdown-PCR is a way to increase the concentration of specific product by using a higher than optimal annealing temperature and then lower the annealing temperature by and negative increment in temperature each cycle. After a couple of these cycles the reaction should contain more specific product and therefore the additional cycles can be run on the optimal annealing temperature without getting any unspecific product. Below is an example of how a touchdowm-PCR can look:
- 98°C for 2:00 - Denaturing and warm up
- 98°C for 0:10 - Denaturing after each cycle
- 65°C for 0:30 - Annealing
- Temperature increment of -0.5°C/cycle
- 72°C for 2:00 - Elongation
- Go to number 2 - repeat 10 times
- 98°C for 0:10 - Denaturing after each cycle
- 55°C for 0:30 - Annealing
- 72°C for 2:00 - Elongation
- Go to number 7 - repeat 30 times
- 72°C for 5:00 - Final extension
- End
