Methods in Molecular Biology • 16 Enzymes of Molecular Biology

42 Landgraf and Wolfes

9O

temp

[*c]

70

50

Primer

Template

Denaturation Annealing

/

time [min]

Fig. 5. Typical PCR temperature protocol.

Polymerisation

3.2.5. Thermocycler
The PCR reaction requires a repeated cycling through temperature
profiles (Fig. 5). Sample volumes of 20-100 lxL are used in small reaction
tubes, which will facilitate the heat transfer from the incubation device.
Temperature control is usually achieved by automated thermocyclers:

• Water bath devices;


• Heating blocks with conventional electrical heating and liquid cooling
  systems; or


• Heating based on other technologies, e.g., Peltier elements or fan heating.
  It is the main feature of the devices to generate fast temperature
  shifts in the range of 30-95°C. Deviations concerning block unifor-
  mity or precision in repeated cycles of more than 1 °C are not tolerable.
  A prerequisite for allele-specific amplification experiments is a highly
  reproducible annealing temperature.
  With regard to the adaptation of published protocols to a specific
  thermocycler, it is very important to consider the way the actual tempera-
  ture is determined and regulated. Three principles can be distinguished: