APPLICATION NOTE

Thermal cyclers

Thermal cyclers: key thermal cycling

concepts and ramp rates
Key specifications of thermal cyclers Temperature (°C)
are often found in the instruments’
literature and on the Web with very
100
little explanation of how the numbers
Block temperature
are derived or used. Ramp rates, curves (all 6 zones)
Average ramp rate
thermal overshoot, and other thermal 90
temperature range
cycling characteristics are key to Maximum ramp rate
temperature range
PCR performance, and are explained 80
in depth here using a variety of
Applied Biosystems™ thermal cyclers
as benchmarks. In addition, ramp 70

rates are tested and compared to Sample temperature

published specifications. 60
curves (all 6 zones)

Ramp rates: maximum vs.

Time
average, and block vs. sample
Temperature cycling is fundamental Figure 1. Real block and sample temperature curves from all six zones during up ramp
to all PCR reactions, and how fast on the Applied Biosystems™ ProFlex™ 96-well PCR system. The dotted lines indicate the
a thermal cycler can ramp between temperature ranges sampled for the maximum ramp rate and the average ramp rate.

temperatures will dictate the overall

speed and duration of the PCR
run. Because of this, thermal cycler is simply expressed by the slope of the curve. A steeper curve represents a
manufacturers publish their ramp rates higher ramp rate, and means that a specific temperature range can be covered
to indicate the change in temperature in a shorter time. The terms “up ramp” and “down ramp” refer to the ramp rates
over time. Ramp rates are typically when heating and cooling, respectively. Of course, a faster-ramping block will
expressed in °C/second. Looking at result in a faster PCR run, so many thermal cycler manufacturers seek to show
this concept graphically on a plot of the highest possible ramp rate. However, the way ramp rates are measured may
temperature vs. time, the ramp rate make them appear to be higher than they really are.
The metal blocks of thermal cyclers must be heated and Overview: sample temperature control
cooled between the steps in PCR such as denaturation, How thermal cyclers attain and measure temperature
annealing, and extension. A commonly published differs, depending on the manufacturer. Often, thermal
specification is the maximum block ramp rate, also cyclers are programmed to achieve temperatures based
known as the peak block ramp rate. The maximum on block temperature alone, without accounting for the
block ramp rate corresponds to the highest achievable temperature of the liquid sample.
block performance. This maximum performance may
only be achieved during a very brief period during the However, just because the block has achieved its desired
ramp. Thermal cycler manufacturers may also publish temperature, or set point, it does not mean that the
the average block ramp rate, which represents the rate sample has achieved the same set point. The proprietary
of temperature change across a longer portion of the temperature control algorithm used by Applied Biosystems
ramp, and is more representative of the thermal cycler’s thermal cyclers helps ensure that temperature set points
performance and speed (Figure 1). are reached quickly and accurately, by accounting for
the sample volume used. This algorithm actually drives
However, just because the block is ramping quickly, it does the block temperature higher than the set point to permit
not mean that the liquid of your PCR reaction (sample) is the sample to reach the set point faster. This is known
ramping just as quickly. It takes time for the heat from the as “block overshoot thermal energy”, depicted in green
thermal cycler’s block to be transferred to the sample. in Figure 2. It is important to note that although the block
Because of this, ramp rates for Applied Biosystems thermal overshoots the set point, the sample should reach the set
cyclers are expressed in terms of average sample ramp point with no overshoot. In addition, the thermal cycler
rate and maximum sample ramp rate. Ramp rates does not begin to count down the time of that step until
that are based on the block temperature alone, without the sample reaches the set point. This helps ensure that
accounting for sample volume, do not reflect the ramp rate the sample spends exactly the amount of time at a specific
of the sample. temperature that you programmed into the thermal cycler.

Applied Biosystems thermal cyclers use a proprietary

algorithm that calculates the temperature of the PCR
sample itself, based on the volume entered. This allows
us to publish sample ramp rates. We also publish block
ramp rates to allow more direct comparisons to other
manufacturers’ thermal cyclers.

Temperature set point

Temperature

Block overshoot thermal energy (green area) is

generated to be transferred to the sample.

Block temperature (sample-driven algorithm)

Sample temperature

Time
Figure 2. Block overshoot thermal energy. Temperature curves for the reaction block (black) and sample (red), and the associated block overshoot
thermal energy (green) during a heating phase that is ideally controlled. The horizontal dotted line indicates the set temperature for this phase.
Our proprietary temperature control algorithm helps ensure Table 1. Measured and published data for Applied Biosystems™
ProFlex™, VeritiPro™, SimpliAmp™, MiniAmp™ Plus, and MiniAmp™
that samples reach a stable set point temperature—within thermal cyclers.
a range of ±0.25°C—without overshoot and associated Max. block rate Max. sample rate
deleterious effects. The algorithm uniquely predicts (°C/sec) at 1 µL (°C/sec)
the temperature and speed of the PCR reaction while Up Down Up Down
accounting for the volume of the PCR reaction and the Block type ramp ramp ramp ramp
thickness of the reaction tube. Sample temperature or Measured data
sample ramp rates are typically not mentioned by other ProFlex 96-well 6.4 5.9 4.6 4.3
thermal cycler manufacturers. ProFlex 3 x 32-well 6.2 5.6 4.4 4.2
VeritiPro 96-well 6.8 6.0 4.9 3.4
Published vs. measured ramp rates SimpliAmp 4.0 3.7 3.1 3.0
Our Applied Biosystems thermal cyclers include information MiniAmp Plus 3.6 3.7 2.7 2.6
on both the maximum block ramp rate and the more MiniAmp 3.5 3.5 2.3 2.0
relevant maximum sample ramp rate. The published data Published data
[1-4] are based on the averages of measured performance ProFlex 96-well 6.0 4.4
data captured during product testing (Table 1). ProFlex 3 x 32-well 6.0 4.4
VeritiPro 96-well 6.0 4.4
Some manufacturers’ published thermal cycler ramp rates SimpliAmp 4.0 3.0
are known to vary significantly from actual measured ramp MiniAmp Plus 3.5 2.7
rates [5]. In a recent comparison of maximum block ramp MiniAmp 3.5 2.2
rates of multiple thermal cyclers, the smallest differences
between the published and measured ramp rates were
observed for Applied Biosystems thermal cyclers (Table 2).
These differences are useful when directly comparing
thermal cycler capabilities.

Table 2. Comparison of average maximum block ramp rates (°C/sec) of thermal cyclers from different manufacturers [5].
ProFlex 96-well ProFlex 3 x 32-well Eppendorf ™ Mastercycler™ X50 Bio-Rad™ C1000 Touch™
thermal cycler thermal cycler thermal cycler thermal cycler
Published Measured Published Measured Published Measured Published Measured
Up Down Up Down Up Down Up Down Up Down Up Down Up Down Up Down
6.0 6.0 6.4 5.9 6.0 6.0 6.2 5.6 5.0 2.3 6.7 3.9 5.0 5.0 3.0 2.4

VeritiPro SimpliAmp Biometra™ TAdvanced Bioer™ LifeECO

thermal cycler thermal cycler thermal cycler thermal cycler
Published Measured Published Measured Published Measured Published Measured
Up Down Up Down Up Down Up Down Up Down Up Down Up Down Up Down
6.0 4.4 6.8 6.0 4.0 4.0 4.0 3.7 6.6 4.3 6.6 4.3 4.2 2.7 4.2 2.7

MiniAmp Plus MiniAmp Bio-Rad™ T100™ Biometra™ TOne

thermal cycler thermal cycler thermal cycler thermal cycler
Published Measured Published Measured Published Measured Published Measured
Up Down Up Down Up Down Up Down Up Down Up Down Up Down Up Down
3.5 2.7 3.6 3.7 3.5 2.2 3.5 3.5 4.0 4.0 2.6 1.6 4.0 3.3 3.4 2.5
Summary
Precise temperature control of thermal cyclers is crucial for
the accuracy and efficiency of PCR experiments. Multiple
factors specific to thermal cyclers, including the actual
thermal cycler ramp rates, sample temperatures, and how
the thermal cycler ensures accurate overshoot calculation,
contribute to the integrity and performance of PCR assays.

Our proprietary algorithm uniquely enables our thermal

cyclers to achieve the correct temperatures of the samples
rather than the blocks; the resulting published ramp rates
help ensure adherence to the programmed protocol and
evaluation of a thermal cycler’s true performance.

References
1. ProFlex PCR System User Guide. P/N MAN0007697, Rev B.0.
2. VeritiPro Thermal Cycler User Guide. P/N MAN0019157, Rev. A.0.
3. SimpliAmp Thermal Cycler User Guide. P/N MAN0009889, Rev. C.0
4. MiniAmp and MiniAmp Plus Thermal Cycler User Guide. P/N MAN0017492, Rev. A.0.
5. Kim YH, Yang I, Bae YS, Park SR (2008) Performance evaluation of thermal cyclers for
PCR in a rapid cycling condition. Biotechniques 44(4):495–505.

Find out more at thermofisher.com/thermalcyclers

For Research Use Only. Not for use in diagnostic procedures. © 2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. C1000 Touch and T100 are trademarks of
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