Bio-Rad C1000 Touch™ Thermal Cycler - Manual (Page 38)

C1000 Touch TM Thermal Cycler Manual
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NOTE: If the primer T
m
values are greater than 4°C apart, the Protocol AutoWriter
uses the lower primer T
m
value + 2°C as a basis for calculating the T
a
value, which
can be further modified by the enzyme and reaction speed selections.
NOTE: The T
a
calculator generates an annealing temperature for standard speed
with iTaq DNA polymerase. When using a different enzyme, the speed settings
automatically adjust the T
a
.
5. Touch the OK button to return to the Protocol AutoWriter screen.
Overview of the Protocol AutoWriter
The Protocol AutoWriter uses standard PCR guidelines that automatically generate cycling
protocols with initial template denaturation and enzyme activation, followed by cycles of
denaturation, annealing, and extension, and then final extension steps. Protocols are based on
user input parameters of target amplicon length, enzyme type, and annealing temperature or
primer sequences.
Protocols generated by the Protocol AutoWriter at various speed settings (standard, fast, and
ultrafast) may result in different product yields, because the software may adjust the annealing
temperature, reduce the total number of protocol steps or GOTO repeats, shorten hold times,
or reduce the temperature differentials between steps to generate these protocols.
The Protocol AutoWriter uses established PCR standards that reference data tables to
produce the final suggested protocols: These are either a standard two-step protocol or a
three-step protocol with a final extension step.
The steps and range of values generated by the Protocol AutoWriter are as follows:
1. Initial hot start activation/denaturation step — this step should be 95–98°C for 30 or
180 sec, depending on the enzyme type and speed setting used.
2. Denaturation step — this step should be either 95 or 98°C for 5–30 sec, depending on
the enzyme type and speed setting used.
3. Annealing step — for this step, the Protocol AutoWriter uses either the primer annealing
temperature (T
a
) at standard speed that is calculated by the T
a
calculator or a value that
has been entered by the user.
• Length of annealing time — this range is 10–30 sec depending on the speed
setting. Two-step protocols combine annealing and extension steps
• The annealing temperature (T
a
) — this temperature is calculated based on
primer characteristics and selected reaction speed. If the iProof enzyme is
selected, 3°C are added to the T
a
value
4. Extension step (for three-step protocols only) — all extension steps are performed at
72°C with the duration based on calculating the times from set times per kbp (for
example, 60 sec/kbp) of the largest amplicon in a size bin, often with some modifications
at the smaller amplicon sizes.
5. Number of repeats — the steps are repeated 25–40 times depending on the speed
selected.
6. Final extension step — this step is performed at 72°C for 1–5 min depending on speed
settings.