Creating Programs
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Choosing a Lid Control Mode
When a sample is heated, condensation on the tube cap or plate cover can take place.
This changes the volume of the sample, the concentration of components, and the
kinetics of the enzymatic reaction. The Hot Bonnet heated lid minimizes condensation
by heating the upper surface of the reaction vessel to a temperature slightly greater
than that of the sample itself. The DNA Engine Tetrad 2 cycler can control lid temper-
ature in three possible ways:
Constant
,
Tracking
, or
Off
.
Constant Mode:
This mode maintains the inner lid surface at a specific temper-
ature regardless of sample temperature. When using constant mode, specify a lid
temperature at least 5°C higher than any temperature used in the protocol.
Note:
We will choose to maintain a constant lid temperature of 100°C in our
example program.
Tracking Mode:
This mode incorproates a temperature offset of the heated inner
lid by a minimum specified number of degrees Celsius in comparison to the
temperature of the sample block. Tracking is useful for protocols with long
incubations in the range of 30–70°C, where it may be undesirable to keep the lid
at a very high temperature. An offset of 5°C above block temperature is adequate
for most protocols.
Off:
No power is applied to the heated lid. In this mode, condensation will occur
at a rate consistent with the incubation temperature and the type of tube or plate
sealant being used. This option is recommended only when using an oil or wax
overlay.
Choosing a Temperature Ramping Rate (Advanced Mode Only)
Fast thermal ramping between incubation steps can often help reduce overall
reaction times by 10% to 30% and may help reduce production of non-specific
products. The Alpha units use multiple zones of thermal control, which allow rapid
ramp rates to be balanced with temperature uniformity.
The DNA Engine Tetrad 2 cycler is capable of ramping temperatures in a range of
–5.0°C to 105.0°C, but no more than 30°C below ambient temperature. The ramp
rate can be as low as 0.1°C/sec, or as fast as 3.0°C/sec. Slower ramp times may be
achieved using a combination of increment and goto steps. Contact Bio-Rad
Technical Support for details. If a ramp rate is not programmed, the default will be at
maximum.
Choosing a Temperature Hold Time
Because of the calculated melting temperature (T
m
) of a DNA hybrid, DNA poly-
merase processivity, and reaction kinetics, it may be possible to generalize
conditions regarding thermal-cycling protocols. However, decisions on denaturation,
annealing or extension hold times will be reaction specific and should be optimized.
A target temperature can be held for as little as 1 second, or up to forever, should a
protocol require an extended incubation period. In graphical programs, the maximum
