© Thermo Hybaid. Issue 1.0, January 2002
44
7.3 Transfer of Protocols from a Block Control Machine to Tube Control
Using the Thermistor Lead Extension
The different modes of control should be considered before transferring protocols directly. E.g.,
consider a temperature cycling protocol consisting of 1 minute at 95°C followed by 1 minute at 65°C,
repeated 30 times. Using block control, the actual sample temperature is at 95°C for just 30 seconds
at each step, a total of 15 minutes overall. In contrast tube control will result in precise one minute
incubations at each step, a total of 30 minutes at the target denaturation temperature. Even though tube
control gives a more accurate representation of the program, transferring such a protocol directly
could result in lower yields because the enzyme is exposed to the high temperature for significantly
longer, thus reducing its activity in later cycles.
The most accurate way to transfer protocols is to use the control tube of the Px2 as a temperature
probe in the block control machine as follows:
1. Set the Px2 to run a single program at a set temperature for an extended time. (e.g. 37
°
C for 4
hrs), ensuring that the heated lid is switched
off
and the instrument is set to
block control
. This
enables the tube thermistor to be used remotely.
2. Connect the thermistor lead extension to the thermistor control tube and plug it into the Px2.
3. Place the thermistor tube probe into the corresponding thermistor probe socket of the Px2, and
locate the tube in a well of the block control machine running the required protocol.
4. After a short equilibration interval, the display on the Px2 will indicate the sample tube
temperature, which should be noted at regular time intervals (e.g. 10 seconds, for a number of
cycles). The actual length of time spent at each of the denaturation, annealing and elongation
stages should be recorded, as well as any temperature overshoot values where the
maximum/minimum temperature exceeds the target temperature or undershoots where the target
temperature is not actually reached.
5. The temperature profile that the samples in the block control machine actually achieve, rather than
simply the block temperature, can be used to program your Px2. Doing this will significantly
reduce the total time required to run a protocol.
7.4 Optimisation of Protocols
The capability of the Px2 programming enables protocol optimisations to be performed very rapidly.
Typically, temperature cycling protocols may consist of three distinct stages:
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