33
Melt Curve Stage
A melt curve is typically performed immediately after the amplification protocol in one
continuous sitting. The purpose is to determine specificity of the PCR reaction and to check
whether nonspecific amplification products, such as primer dimers, were formed. Refer to
Section 1.4 Melt Curve Analysis for more details.
ADD PAUSE
You may also select
Add Pause
to a certain step. When a pause is implemented, the reaction
will be held for an indefinite amount of time at a set temperature (figure 5.10). Once an
experiment reaches the pause stage, the
In Progress
time will display
00:00:00.
Select the
Resume
icon on the bottom of the page layout’s palette to continue the experiment (Figure 4.9).
Figure 4.9
AUTODELTA
The
Autodelta
option is used for Touchdown PCR (TD-PCR) and can be applied only to the cycling
stages on the protocol setup page. Touchdown PCR can be incorporated as part of any standard
PCR to increase the specificity, sensitivity and yield of the PCR reaction. It increases the
likelihood that your target of interest is amplified in cases where there may be significant mis-
priming. It is usually applied if your primers tend to bind non-specifically at temperatures close
(but lower) to the annealing temperature of the correct positions.
TD-PCR involves a cycling program that gradually decreases the annealing temperature over
the course of successive cycles until the calculated melting temperature (T
m
) of the primers is
reached. Typically, the initial annealing temperature is set approximately 10
°
C higher than the
expected Tm of the primers.
