Bibby Sterilin Techne 3Prime, Руководство оператора

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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 loose 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.
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