Eppendorf Mastercycler Gradient - RAPD-PCR Fingerprinting Example

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7 Programming
7.6.5 PCR "Fingerprinting" (RAPD-PCR)
Variable heating and cooling rates, which can be programmed as desired with each temperature command,
enable the temperature-control speed to be aligned to the temperature.
With the RAPD-PCR process or with PCR with A/T-rich primer/template hybrids, it may be necessary to heat
up carefully after annealing.
In the example given, the elongation temperature is attained at a speed of 1 °C/s.
Temperature-control speeds, which may be as slow as the user desires, can be selected by means of program
loops, with which a gradual change in temperature is obtained via temperature increments.
In the following example, the elongation temperature is raised slowly to 72 °C after annealing with the aid of a
program loop.
RAPD-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 55° 15 sec
4 Elongation 72° 30 sec
Heating rate R=1 ° /s
5 Number of cycles go to 2 Rep 29
6 Final elongation 72° 2 min
7 Cooling and storage Hold 20° enter
End end
RAPD-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 55° 15 sec
4 Elongation 56° 7 sec
Temperature increment
Heating rate
+1 °
R=0.3 ° /s
5 Number of cycles go to 4 Rep 16
6 Number of cycles go to 2 Rep 29
7 Final elongation 72° 2 min
8 Cooling and storage Hold 20° enter
End end