Thermo Hybaid PX2 Operator's manual
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THERMO HYBAID
Px2 Thermal Cycler
USER INSTRUCTION MANUAL
Head Office
Thermo Hybaid
Action Court
Ashford Road
Ashford
Middlesex TW15 1XB
Telephone: +44 (0) 1784 425 000
Facsimile: +44 (0) 1784 248 085
Internet: www.thermohybaid.com
STOCK CODE:
HB-PX2-MAN
FS31999
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© Thermo Hybaid. Issue 1.0, January 2002
ii
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© Thermo Hybaid. Issue 1.0, January 2002 iii
Px2 THERMAL CYCLER
PCR License Registration
Authorised Thermal Cycler
This instrument, Serial No. ................................................ is an Authorised Thermal Cycler. Its
purchase price includes the up-front fee component of a license under United States Patent Nos.
4,683,195, 4,683,202 and 4,965,188, owned by Roche Molecular Systems, Inc., and under
corresponding claims in patents outside the United States, owned by F. Hoffmann-La Roche Ltd,
covering the Polymerase Chain Reaction (“PCR”) process, to practise the PCR process for internal
research and development using this instrument. The running royalty component of that license may
be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents. This
instrument is also an Authorized Thermal Cycler for use with applications licenses available from
Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in
accordance with the label rights accompanying such reagents. Purchase of this product does not
itself convey to the purchaser a complete license or right to perform the PCR process. Further
information on purchasing licenses to practice the PCR process may be obtained by contacting the
Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California
94404, USA.
No rights are conveyed expressly, by implication or estoppel to any patents on real-time methods,
including but not limited to 5' nuclease assays, or to any patent claiming a reagent or kit.
Applied Biosystems does not guarantee the performance of this instrument.
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© Thermo Hybaid. Issue 1.0, January 2002 iv
Key Pad
Chassis
Main Body
Stabilising Feet
Heated Lid Height Selector
Heated Lid
Large LCD Screen
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© Thermo Hybaid. Issue 1.0, January 2002 v
Px2 THERMAL CYCLER
Warranty
Thermo Hybaid guarantees that the Px2 Thermal Cycler you have received has been thoroughly
tested and meets its published specification.
This warranty is valid for 24 months only if the product and functions have been used according to
the instruction manual. No liability is accepted for loss or damage arising from the incorrect use of the
Px2 Temperature Cycling system. Thermo Hybaid's liability is limited to the repair or replacement of
the unit or refund of the purchase price at Thermo Hybaid's option. Thermo Hybaid is not liable for
any consequential damages.
The tube thermistor assembly supplied with your Px2 unit is guaranteed for 90 days.
Thermo Hybaid reserves the right to alter the specification of the Px2 without prior notice. This will
enable us to implement developments as soon as they arise.
The Thermo Hybaid Px2 is for research use only.
Read the Instruction Manual carefully before using the Px2 to ensure that you obtain the
best possible results from the machine.
NB: The Px2 should only be used by suitably qualified and trained people. If the Px2 is
not used as specified in this Manual, the protection provided by the equipment may
be impaired.
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Px2 THERMAL CYCLER
Contents
PCR LICENSE REGISTRATION.........................................................................................iii
Px2 DIAGRAM...................................................................................................................... iv
WARRANTY...........................................................................................................................v
CHAPTER 1: INTRODUCTION ......................................................................................1
1.1 Overview............................................................................................1
1.2 Px2 System.........................................................................................1
The 0.2ml Standard and Gradient Block Module ...................................1
The 0.5ml Standard and Gradient Block Module ...................................1
The Flat Block Module .........................................................................1
The 384 Well Block Module ................................................................2
Temperature Control Methods..............................................................2
Heating and Cooling..............................................................................2
Programming and Operation..................................................................3
Pre-set Programs..................................................................................3
Advanced Edit –Temperature Ramping ................................................3
Advanced Edit –Time/Temperature Increment ......................................3
Advanced Edit –Gradient (optional) .....................................................4
Calibration of your Px2 .........................................................................4
1.3 The Heated Lid..................................................................................4
Operating the Lid..................................................................................4
1.4 Input / Output Sockets.......................................................................6
1.5 Support Services................................................................................6
CHAPTER 2: SAFETY PRECAUTIONS..........................................................................7
2.1 General Safety Precautions...............................................................7
2.2 Symbols and Conventions..................................................................8
CHAPTER 3: UNPACKING & INSTALLATION...........................................................9
3.1 Unpacking...........................................................................................9
3.2 Packing List........................................................................................9
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3.3 Installation........................................................................................10
Choosing a Location...........................................................................10
Block Installation and Removal............................................................10
Thermistor Positioning.........................................................................11
Use in Cold Rooms.............................................................................11
CHAPTER 4: OPERATING INSTRUCTIONS.............................................................13
4.1 Initial Start Up..................................................................................13
4.2 Main Menu......................................................................................13
4.3 Operating Keys................................................................................14
4.4 Set Up Functions ..............................................................................14
Clock.................................................................................................14
Power Failure Resume ........................................................................15
4.5 Gradient Calculation........................................................................15
4.6 Running a Program..........................................................................16
4.7 Manual Operation............................................................................16
4.8 Programming Function.....................................................................17
4.9 Loading Samples and Heated Lid Operation.................................18
CHAPTER 5: PROGRAMMING THE Px2 ..................................................................19
5.1 Introduction......................................................................................19
5.2 Directories........................................................................................19
Creating a New Directory...................................................................19
Editing a Directory Name....................................................................20
Erasing an Existing Directory...............................................................20
5.3 Programs ..........................................................................................21
Creating a New Program....................................................................21
Editing an Existing Program.................................................................22
Viewing an Existing Program...............................................................24
Copying Programs ..............................................................................24
Erasing an Existing Program................................................................25
5.4 Advanced Edit Features..................................................................25
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Time Advanced Worked Example.......................................................26
Temperature Advanced Worked Example...........................................27
Changing Ramp Rates.........................................................................28
5.5 Gradient Feature..............................................................................28
Gradient Programming Worked Example.............................................28
Invalid Gradient Parameters ................................................................29
Gradient Calculator Function...............................................................29
CHAPTER 6: RUNNING A PROGRAM ON THE Px2................................................31
6.1 The Run Option................................................................................31
6.2 Running a Program on the Px2 –Short Instructions......................31
6.3 Run Screens .....................................................................................32
Text Screen........................................................................................32
Power Failure Screen..........................................................................33
End of Run Screen..............................................................................33
Error Screen.......................................................................................33
6.4 Temperature Control Options .........................................................33
Active Tube Control (ACTIVE TUBE)...............................................33
Extension Lead for Tube Thermistor....................................................34
Simulated Tube Control (SIM TUBE).................................................35
Simulated Plate Control (SIM PLATE) ...............................................35
Simulated Slide Control (SIM SLIDE) ................................................36
Block Control.....................................................................................37
6.5 Heated Lid Preheat..........................................................................37
Heated Lid -Automatic Start..............................................................37
Heated Lid -Manual Start ..................................................................37
No Heated Lid ...................................................................................38
6.6 Hold and Pause Function.................................................................38
6.7 Aborting Programs...........................................................................38
6.8 Program Completion........................................................................39
CHAPTER 7: TROUBLESHOOTING............................................................................41
7.1 Programs Using ACTIVE Tube Control.........................................41
Check the Volume in the Tube Thermistor...........................................41
Check the Location of Thermistor within the Control Tube...................41
Check the Fit of the Tube in the Block.................................................41
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Check Through Program.....................................................................41
7.2 Programs Using SIM Temperature Control...................................42
Simulated Tube...................................................................................42
Simulated Plate...................................................................................42
Simulated Slide...................................................................................42
Power Failure during a Run.................................................................42
7.3 Transfer of Protocols from a Block Control Machine
to a Tube Control Machine Using the Thermistor
Lead Extension.................................................................................43
7.4 Optimisation of Protocols.................................................................44
Denaturation.......................................................................................44
Annealing............................................................................................44
Extension............................................................................................45
CHAPTER 8: MAINTENANCE......................................................................................47
8.1 General Cleaning.............................................................................47
8.2 Decontamination..............................................................................47
8.3 Protection to the User......................................................................48
8.4 Protection of the Instrument............................................................48
Fuses..................................................................................................48
8.5 Tube Thermistor Care .....................................................................48
CHAPTER 9: TECHNICAL SPECIFICATIONS &
ORDERING INFORMATION ...............................................................51
APPENDIX I: RECOMMENDED CONSUMABLES...................................................57
APPENDIX II: SEALING SYSTEMS: THE OMNISEAL TD MAT & TD TAPE........59
APPENDIX III: IN SITU BLOCK APPLICATION NOTES ............................................63
APPENDIX IV: GLOSSARY OF TERMS.........................................................................65
APPENDIX V: PRE-SET PROGRAMS ON THE Px2....................................................69
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CHAPTER 1
Px2 THERMAL CYCLER
Introduction
1.1 Overview
Px2 is the essence of fast, accurate licensed thermal cycling. Px2 features high capacity, high speed
and sub-ambient blocks to perform oil-free thermal cycling with excellent dynamic uniformity and
precision control of sample temperature.
1.2 Px2 System
The Px2 System comprises a Control Chassis and an Interchangeable Block Module. Each control
chassis can operate one block module which can be changed according to the needs of the sample
format. There are six types of block module:
The 0.2ml Standard and Gradient Block Module
This block can hold any of the following:
•1 x OmniUtra 96 well skirted polycarbonate plate
•1 x OmniFast 96 well skirted polypropylene plate
•1 x OmniTube 96 single piece polypropylene plate
•96 x 0.2ml OmniStrip tubes
•96 x 0.2ml individual OmniTubes
The 0.5ml Standard and Gradient Block Module
This block can hold the following:
•48 standard 0.5ml individual OmniTubes
The Flat Block Module
This block accommodates up to four standard microscope slides.
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The 384 Well Block Module
This block accommodates 1 x OmniFast 384 well skirted polypropylene plate.
Temperature Control Methods
The temperature cycling blocks for the system are specially coated precision-machined aluminium.
This ensures excellent contact between the tubes and the block, enabling rapid and accurate heat
transfer from the block to the samples.
1. Active Tube Control
Accurate sample temperature control in 0.2ml and 0.5ml block types is achieved by Thermo Hybaid's
Active Tube Control software. A tube thermistor probe monitors the temperature within a dummy
sample tube and this information is fed back to precisely control the block temperature to achieve the
optimum cycling profile.
2. Simulated Tube/Plate Control
Alternatively, Simulated Tube Control or Simulated Plate Control may be used for reactions
when it is not appropriate to use the tube thermistor probe, for example in very small (<20µl)
reactions or reactions in 96 Well or 384 Well OmniPlates or racked OmniTubes. The temperature
control algorithm is similar to active tube control, but is based on calculated values for the sample
temperature rather than values fed back by the tube thermistor.
3. Simulated Slide Control
For the in situ module Simulated Slide Control should be selected. This operates on a similar
principle to Simulated Tube Control, but in this case the algorithm is based on achieving the set
temperatures at the top surface of a standard glass microscope slide (1mm thick).
Heating and Cooling
The Px2 sub-ambient blocks are built to proven designs, providing an accurate and reliable thermal
cycling system.
The sub-ambient aluminium block is heated and cooled by the latest in Peltier technology. With
proven durability, the block modules excel in performing applications such as RAPDs and Differential
Display, which require cycling temperatures close to ambient. The block modules will control the
temperature of the samples from 20°C to 99°C for cycling reactions in all reaction formats. In
addition, static incubation steps may be performed as low as 4°C.
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Programming and Operation
The combination of new keypad design and large screen display ensure user friendly programming and
operation. During programmed operation the display screens provide comprehensive information
relating to your protocol including sample temperature, number of cycles completed, estimated time
for completion, etc.
The Px2 can thus be programmed to perform all types of temperature controlled reactions, from
simple one step incubations, to complex multi-step temperature cycling protocols and temperature
gradient experiments*. Three additional temperature control software options are available to the user
in the Advanced Edit Menu: Temperature Ramping, Time Increment and Temperature Increment.
The unit has program space for up to 99 full cycling protocols including the pre-set protocols.
* With the gradient interchangeable block option.
Pre-set Programs
The Px2 is supplied with ten (non-editable) pre-set protocols, stored as programs 90-99 in the
F:THERMO directory, enabling users to run experiments immediately. These pre-set protocols cover
the most common thermal cycling techniques, and can be used to create customised protocols by
editing (see Appendix V).
Advanced Edit -Temperature Ramping
The temperature ramping (Ramp Rate) enables the rate of change of sample temperature (°C/second)
to be artificially slowed down.
Under normal circumstances the temperature cycling times are very rapid, which minimises non-
specific reactions. The rate of sample temperature change during temperature cycling is controlled so
that it is as fast as possible without affecting the block uniformity and accuracy. In some instances, it
may be advantageous to limit the rate of change of temperature, e.g. to allow limited extension of short
or degenerate primers between primer annealing and DNA synthesis steps to stabilise the
primer/template duplex.
Advanced Edit -Time/Temperature Increment
These features enable the time interval and/or the temperature of a specified programmed step to be
increased or decreased with successive temperature cycles. It may be advantageous to increment the
extension time interval to compensate for deterioration of enzyme activity in later cycles. Alternatively,
temperature decrements can be used, e.g. in Px2 cycling reactions where the annealing temperature is
decreased with successive cycles.
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Advanced Edit –Gradient (optional)
For rapid optimisation of primer annealing parameters the Px2 gradient feature should be used. A
maximum temperature gradient of 15°C can be programmed across the 96 well block.
Calibration of your Px2
Every machine is calibrated using miniature thermistor probes located in tubes or attached to
microscope slides. These are placed at several block positions simultaneously, ensuring the required
temperatures and incubation times achieved are identical in all samples.
1.3 The Heated Lid
The heated lid enables the running of temperature cycling protocols without the need for paraffin or
mineral oil overlays. Such vapour barriers are normally required to prevent evaporation before thermal
cycling is complete. This system is designed to be compatible with most reaction sample formats, i.e.
0.5ml, 0.3ml, 0.2ml tubes and 96/384 well plates. In most cases, the experimental protocol is largely
unchanged when switching to an oil-free format.
The heated lid operates by positioning a heated plate in contact with the top of the reaction tubes or
wells. This heated plate then heats the air temperature at the top of each reaction mixture to a
temperature that is permanently higher than the sample temperature. This elevated air temperature,
relative to the sample temperature, minimises evaporation so reducing condensation of the reaction
mixture as it is repeatedly heated and cooled. Condensation on the walls of the reaction vessels below
the level of the block is a feature of block based thermal cyclers. The level of this condensation is very
small and will not affect the reaction in most cases. In very small volume reactions it may however be
necessary to use an oil overlay.
Operating the Lid
Opening the Lid
The lid is opened by turning the knob so that the 'open lid' icon is aligned to the front of the unit (see
Figure 1.1). This releases the catches to raise the lid and expose the block recess.
Closing the Lid
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To close, gently lower the lid. At this point a ‘click’ should be heard as the catches are engaged. This
action lowers the heated lid onto the samples and closes the block, ensuring a tight contact.
The pressure; exerted by the lid can be adjusted for tubes or plates by rotating the knob so that the
relevant icon is aligned to the front of the unit (see Figure 1.1). It is defaulted to the tube pressure, for
the plate pressure turn the knob clockwise until the plate icon is aligned with the front of the unit.
Careful consideration should be taken in selecting which setting should be used. When using a 0.2ml
block, any consumable (including compression pad) whose height exceeds 10mm above the rim of the
block should be used on tube setting whether it is a plate or not. With a 0.5ml blocks, any
consumable (including compression pad) whose height exceeds 5mm above the rim of the block
should be used on tube setting whether it is a plate or not. Please note that the tube thermistor
must be removed before selecting the plate mode.
Figure 1.1: Open Lid Icon Tube Icon Plate Icon
The temperature of the heating plate is set at 120°C (max. surface temperature) with power control
effected by the control chassis.
The heated plate inside the lid is switched on and off from within the program. The lid operates once a
program is activated, with a heating time of typically 2-3 minutes before cycling commences. During
this heating time, the block is automatically cooled to 4°C to maintain sample integrity. The block can
be operated with or without the lid being switched on (the lid is automatically deactivated in units fitted
with flat block modules).
WARNING: Both the tops of reaction vessels and the surfaces of the heated lid
assembly (in particular the inner surfaces), can become very hot during normal
operation. Touching the surfaces can cause burns. Do not touch the heated plate
without safety gloves.
1.4 Input / Output Sockets
•The Px2 has a power input socket located on the back of the unit
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•Communications to the unit may be facilitated by the RS232 / 485 Comm Port via 9 way D-Type
connector. The communication protocol may be selected using the slide switch adjacent to the
connector (located on the base of the unit). The position closet to the front of the unit
corresponds to the RS232, whilst the position closest to the back of the unit corresponds to
RS485.
1.5 Support Services
The Px2 has been designed for reliability and for ease of maintenance. Thermo Hybaid continues to
offer full service and technical support for all its products.
Thermo Hybaid Head Office:
Tel: +44 (0) 1784 425 000
Fax: +44 (0) 1784 248 085
Alternatively, appropriate contact details for your local Thermo Hybaid subsidiary or authorised
distributor are provided on our website: www.thermohybaid.com
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CHAPTER 2
Px2 THERMAL CYCLER
Safety Precautions
2.1 General Safety Precautions
1. The Px2 should only be used by suitably qualified and trained personnel. The unit should only be
used for its intended purpose in accordance with the instructions and safety warnings contained
within this manual.
2. Before use, ensure that the unit has been set to the appropriate mains voltage.
3. The Px2 is a class 1 appliance. To minimise the risk of electric shock, the unit must be connected
to a protective earth via the supplied mains cord.
4. Replacement fuses must be of the correct rated current, voltage and type.
5. Do not operate the unit in an explosive environment.
6. Do not operate the unit if it appears to be damaged or if a liquid or foreign object has entered the
enclosure. Disconnect from the mains supply and contact an authorised service centre.
7. Do not attempt to dismantle the unit. To avoid the risk of personal injury and to ensure that the
safety features of this unit are maintained, servicing should only be carried out by authorised
service personnel.
8. The tops of the reaction vessels and the surfaces of the heated lid assembly (in particular the inner
surfaces) can become very hot during normal operation. Touching the surfaces can cause burns.
Do not touch the heated plate without safety gloves.
9. When positioning the unit ensure that there is no restriction to the power inlet. Special care should
be taken not to obstruct the vent underneath the unit, for example loose Benchcote or sheets of
paper.
10. When installing/removing the interchangeable block
•Ensure that the power to the instrument is turned off.
•The Block Heat Sink may be hot after use. Use gloves when changing blocks if the unit
has been used recently.
11. This product is fitted with RFI suppression circuitry. Testing of the electrical insulation should only
be carried out using a DC voltage. For more information please contact Thermo Hybaid.
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2.2 Symbols & Conventions
The Px2 has been designed for safe operation. The following symbols appear on the unit and their
meanings should be noted.
I Indicates the ON position of the main power switch.
O Indicates the OFF position of the main power switch.
Consult the manual for further information.
Consulter les documents d’accompagnement.
WARNING: Indicates a heating hazard. Proceed with caution to avoid burn injury.
ATTENTION:Surface chaude.
SAFETY NOTE: This symbol indicates high voltage. Risk of electric shock.
AVERTISSEMENT:Risqué de choc éléctrique.
In addition, the following conventions are adopted in the manual in respect of indicating safety hazard:
SAFETY NOTE: indicates a potentially hazardous situation, which could result in
death or serious injury.
WARNING: indicates a potentially hazardous situation, which could result in minor
or moderate injury to the user or damage to the instrument.
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CHAPTER 3
Px2 THERMAL CYCLER
Unpacking & Installation
3.1 Unpacking
Before unpacking the Px2, please make sure that the outer packaging is undamaged.
After unpacking, ensure all packaging and fixtures are retained. Should there ever be a requirement to
move the unit, it should always be transported in the original packing to avoid damage. Thermo
Hybaid cannot accept responsibility for any damage incurred if the unit is incorrectly packed and
transported.
N.B. If the unit is transported or stored in conditions of high humidity it must be allowed to
stabilise at normal ambient temperature before powering up the unit.
3.2 Packing List
1. Px2 Control Chassis
2. Px2 Block Module (optional)
3. User Instruction Manual
4. Mains Lead
5. Control Thermistor Probe (0.2ml and 0.5ml gradient and standard block modules)
6. Control Thermistor Probe Extension Lead (0.2ml and 0.5ml gradient and standard block
modules)
7. Specific Consumable Pack
8. RS232 Lead
NB: The In Situ Flat Block Module includes a humidity chamber.
If any item is missing or damaged, contact the UK Service Department, or your local Thermo Hybaid
office/authorised distributor.
Ensure any padding between the plate inside the heated lid and the block is removed before using the
instrument.
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