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11. Some frequently asked questions
Q1 What is the ‘Pause’ function at the start of the program used for?
A1 Some users prefer to preheat the heated lid before placing the samples into the
unit. The pause feature is used to stop the unit after the 4 minut e heated lid
preheat step. It will also sound an audible alarm indicating that the machine is
ready for the sample tubes or pla te to be added. Pressing the “Pause” key
followed by the “Enter” key will commence the remaining program.
Q2 Why do you have to press some keys twice to make the unit do what you want
it to do?
A2 With many of the program f eatures there is a fail-safe function which asks if
you are sure you want to perform a particular action. You are required to press
one key to select a function followed by a second to accept it.
Q3 What is ‘Hot Start’?
A3 A “hot start” is used for reducing non-specific product formation during the
first cycle of amplification. The ‘Hot S tart’ programming step is used to pause
the instrument at a specific temperature, typically around 70ºC, after the initial
template denaturation. The reason is to allow the manual addition of
unmodified Taq DNA polymerase which may loose activity if added during the
initial 5min denaturation. Heat-activated Taq or Hot Start enzy mes do not
require this step.
Q4 What is the incremental function? (See page 24).
A4 Incremental timing and temperature are used to increase or decrease either the
time or temperature incrementally over the number of cycles in a stage.
Incrementation of extension time is used with ‘Long PCR’ which is when large
template fragments are to be amplified (e.g. 27kb lambda DNA, 40kb ge nomic
DNA).
Decremental temperature is used for protocols such as ‘Touchdown PCR’
where one starts with a high annealing temperature in the first cycle and
gradually decreases the temperature over the numbe r of cycles in the stage.
This ensures that only the specific product is amplified.
Q5 The heat-up ramp-rate in the specification is 3.6°C/second yet I can only
manually set it to 2.0°C/second. Why is this?
A5 If you set the ramp-rate to max the block will heat at 3.6°C/second the unit sets
its own control parameters. If you program a ramp-rate the control parameters
reduce the ramp-rate. This is a feature of the software and not the actual ramp-
rate.