Team:DTU-Denmark/Methods/PCR-touchdown

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Revision as of 19:30, 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
- Negative temperature increment of 1°C/cycle
72°C for 2:00 - Elongation
Go to number 2 - repeat 5 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 10 times
72°C for 5:00 - Final extension
End

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-hbgfs
+== 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
+#* Negative temperature increment of 1°C/cycle
+# 72°C for 2:00 - Elongation
+# Go to number 2 - repeat 5 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 10 times
+# 72°C for 5:00 - Final extension
+# End