Techne 3PrimeG - Manual (Page 50)

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FAQS
Q1 How do I adjust the pressure of the heated lid for my tubes or plates?
A1 For thermal cyclers with lid adjustment knobs, rotate the knob anticlockwise until there is
no pressure on the consumable, then close the lid and latch it. To obtain the correct
pressure gently rotate the knob clockwise until it is possible to just feel the pressure being
applied. Finally, rotate the knob a further quarter of a turn; the lid is now at the correct
pressure for use with the consumable. Ensure that all consumables used in the block are
of the same height and are spread evenly across the block. Insert empty “dummy” tubes if
necessary to spread the pressure of the heated lid evenly.
Q2 What is the '”Pause” function at the start of the program used for?
A2 Some users prefer to preheat the heated lid before placing the samples into the unit. The
pause feature is used to pause the unit after the 2 minute heated lid preheat step.
Q3 What is “Sample Cooling”?
A3 Sample cooling can be applied during the lid pre-heat phase. This maintains the block at
4ºC while the lid is heating to temperature and avoids any increase in sample temperature
due to the heated lid before the thermal cycling program begins.
Q4 What is "Hot start"?
A4 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 lose activity if added during the
initial denaturation step. Heat-activated Taq or Hot Start enzymes do not require this step.
Q5 What is the incremented time and temperature function for?
A5 Incremented/decremented time 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). Decremented
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 in the stage there is a
gradual decrease in the temperature. This ensures that only the specific product is
amplified.
Q6 What is a gradient thermal cycler?
A6 Gradient blocks enable a particular temperature step in a protocol to be programmed 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. The set temperature is in
the middle of the 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 approximately 55°C to 65°C from
left to right.
Q7 Why is a temperature gradient required?
A7 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 ( 3 PrimeG) allows the testing of up to 8 different
annealing temperatures simultaneously.