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FREQUENTLY ASKED QUESTIONS
Q1
What is "Hot start"?
A1
The Hot Start step is used to pause the machine at a specific temperature, typically around 70°C,
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 enzymes do not require this step.
Q2
What is the incremental function? (See page 36).
A2
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 genomic DNA).
Decremental temperature is used for protocols such as 'Touchdown PCR' where the first cycle
starts with a high annealing temperature and over the number of cycles there is a gradual
decrease in the temperature. This ensures that only the specific product is amplified.
Q3
What is a gradient thermal cycler?
A3
Gradient blocks enable a particular temperature step in a protocol to be altered so that the
temperature varies across the block. By specifying a temperature and the gradient to be applied,
each column will achieve a different temperature, with the set temperature in the middle block,
the lowest on the left and the highest on the right. The gradient is the total difference across the
block, for example if the set temperature was 60°C and the gradient 10°C then the
temperatures would range from 55°C to 65°C from left to right.
Q4
Why is a temperature gradient required?
A4
The annealing temperature of the specific primers used in DNA amplification often requires
optimisation. Instead of running multiple experiments with different annealing temperatures the
gradient thermal cycler allows the testing of 12 different annealing temperatures simultaneously.
If you require further technical or applicational assistance please contact Bibby Scientific Ltd at:
[email protected].
For servicing information please contact: [email protected].
We are continually striving to improve our thermal cyclers and software. If you have any comments and
suggestions on how we can do things better please send them to us at:
[email protected]
TC512 Techne Manual 19/12/08 3:30 PM Page 53
