Eppendorf Mastercycler Gradient - Touch Down PCR Example; Long PCR Example

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7 Programming
7.6.3 Touch Down PCR
The temperature increment, which can be programmed as desired with every temperature command, allows
the temperature to be reduced or increased by a defined value with every cycle.
Touch Down PCR can be used to increase the specificity of the PCR by changing the the annealing
temperature in succession from higher to lower temperatures.
In the example given, the annealing temperature is reduced by 1 °C per cycle for the first 15 cycles, until 50 °C
has been attained. At this annealing temperature,14 additional cycles are carried out.
7.6.4 Long PCR
The time increment, which can be programmed as desired with every temperature command, enables a
defined extension of the lag time at a specific temperature for each cycle.
With Long PCR, this function can be used to amplify long fragments for a successive extension of the
elongation.
In the example given, the elongation time is extended by 10 seconds for each cycle, which means that, for the
30th cycle, the elongation time is 290 seconds longer than for the first cycle.
Touch Down PCR
Temperature control of tube Control tube
Temperature control of lid lid 105°
Nowait auto
1 Initial denaturation 94° 2 min
2 Denaturation 94° 15 sec
3 Annealing 65° 15 sec
Temperature increment –1 °
4 Elongation 72° 30 sec
5 Number of cycles go to 2 Rep 15
6 Denaturation 94° 15 sec
7 Annealing 50° 15 sec
8 Elongation 72° 30 sec
9 Number of cycles go to 6 Rep 13
10 Final elongation 72° 2 min
11 Cooling and storage Hold 20° enter
End end
Long PCR
Temperature control of tubes Control tube
Temperature control of lid lid 105°
Nowait auto
1 Initial denaturation 94° 2 min
2 Denaturation 94° 15 sec
3 Elongation 68° 10 min
Time increment +0:10 sec
4 Number of cycles go to 2 Rep 29
5 Final elongation 72° 15 min
6 Cooling and storage Hold 20° enter
End end