Techne 3PrimeG - Manual (Page 21)

20
INCREMENTED/DECREMENTED TIME AND TEMPERATURE
The software allows the user to either increment or decrement the time and/or temperature of a step
within a cycling stage. These features are used with applications such as touchdown PCR where the
annealing temperature is gradually decreased during the cycling process and long PCR, where due to
the size of the product, long annealing/extension times are required and the extension time is
increased by 15 to 20s per cycle during the final stages of the reaction.
GRADIENT ( 3 P RIME G ONLY)
The gradient feature of the 3 PrimeG can be useful in optimising the annealing conditions for primers. A
gradient can be set around a temperature in any step of a program. The set temperature is the
temperature in the middle columns of the block and the gradient is the variation at the two extremes of
the block; the left hand columns being the coolest and the right hand columns the hottest.
The maximum temperature gradient range which can be set is 14ºC and the minimum is 1ºC; the
lowest temperature is 30ºC and the hottest temperature is 80ºC. Examples showing approximate
temperatures are given in the table below:
Lowest temperature
(ºC)
Set temperature
(ºC)
Highest temperature
(ºC)
Gradient
(ºC)
50 55 60 10
30 37 44 14
66 73 80 14
A gradient calculator is included in the Settings module so the user can obtain an accurate estimation
of the temperature of each column during the gradient stage. The temperatures for any particular
gradient are calculated from an algorithm stored in the block firmware.
OLIGONUCLEOTIDE T
M
CALCULATOR
One of the most important parameters in a PCR is the annealing temperature of the reaction. This can
be determined theoretically using various software programmes to calculate the melting temperature
(T
m
) of the primers.