Bio-Rad C1000 Touch™ Thermal Cycler - Manual (Page 39)

C1000 Touch TM Thermal Cycler Manual
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The Protocol AutoWriter uses information about the reaction to automatically write a protocol.
This feature enables the user to enter the following information about the PCR experiment:
• Primers — enter the reaction T
a
for the primers being used. If the T
a
is not known,
enter the primer sequence in the T
a
calculator so that the Protocol AutoWriter
calculates this value
NOTE: The T
a
is adjusted from the primer melting temperature (T
m
) information that
is based on the selected enzyme and the protocol speed selected.
• Amplicon length — enter the expected length of the PCR product
• Enzyme type — enter the DNA polymerase enzyme (iTaq, iProof, or Other)
If an enzyme other than iTaq or iProof DNA polymerase is to be used, enter additional
information including the hot-start activation time and the final extension time
• Desired run speed — select a reaction speed (standard, fast, or ultrafast).The
Protocol AutoWriter optimizes the protocol depending on the selected speed setting
The total run time is determined by the number of steps and cycles, the incubation time at
each step, and the time it takes to reach uniformity at the target temperature.
To reduce the overall run time, the Protocol AutoWriter reduces one or more of the following:
• The total number of protocol steps (for primers with high T
m
values, the annealing
and extension steps can be combined into one step)
• The number of cycles
• The hold time in each temperature step
• The ramp time between steps (by reducing the temperature change from one step to
the next)
For example, the Protocol AutoWriter may shorten a protocol by:
• Changing the initial template denaturation and enzyme activation step from 95°C for
3 min to 98°C for 30 sec hold time in each temperature step
• Changing the denaturation step in each cycle from 95°C for 30 sec to 92°C for 1 sec
• Combining the annealing and extension steps into a single step at 70°C for 20 sec
The T
a
Calculator
The T
a
calculator calculates the T
m
value for each primer as well as the T
a
value for the
protocol at standard speed.
The T
a
for the protocol is based on the average primer T
m
values with the following applied:
• If the difference between the primer T
m
values is > 4°C, the T
a
= (lower of the two
primer T
m
values + 2) – 4°C
• If the difference between the T
m
values is < or = 4°C, the T
a
= (average of the
primer T
m
values) – 4°C
For each primer, the T
a
calculator uses the base pair counting method for sequences of 14
pairs or less.
• T
m
= ((w*A + x*T) * 2) + ((y*G + z*C) * 4)
where w, x, y, and z are the number of the bases A, T, G, and C in the sequence,
respectively.
For sequences longer than 14 bp, the nearest neighbor method is used: