User Manual - Hybaid - PXE - HBPXE Thermal Cycler

Analyze Detect Measure Control™

PxEThermal Cycler

HBPXEMAN Rev. 9 8 January 2004

Operation Manual

HBPXEMAN Rev. 9 8 January 2004 of 70

PCR License RegistrationAuthorized Thermal Cycler

This instrument, Serial No. ................................................ is an Authorized 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. Hoffman-La Roche Ltd, covering the Polymerase Chain Reaction (PCR) process, to practice 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 estroppel 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.

FS31999


WarrantyThermo Electron Corporation guarantees that the PxE Thermal Cycler you have received has been thoroughly tested and meets its published specification.

This warranty is valid for 12 months from date of original retail purchase only if the product and functions have been used according to the user instruction manual.

This warranty period can be extended to a total of 24 months (free of charge) by completing the warranty registration card supplied with the instrument, also available online at www.thermo.com/warrantylog.

No liability is accepted for loss or damage arising from the incorrect use of the PxE Thermal Cycler. Thermo's liability is limited to the repair or replacement of the unit or refund of the purchase price at Thermo's option. Thermo Electron is not liable for any consequential damages.

Thermo Electron reserves the right to alter the specification of the PXE Thermal Cycler without prior notice. This will enable us to implement developments as soon as they arise.

The Thermo Electron Corporation PxE is for research use only.

Read the Instruction Manual carefully before using the PxE Thermal Cycler to ensure that you obtain the best possible results from the machine.

* Excludes tune thermistor accessory, which carries a warranty for 90 days only.

NOTE: The PxE Thermal Cycler should only be used by suitably qualified and trained people. If the PxE Thermal Cycler is not used as specified in this Manual, the protection provided by the equipment may be impaired (see Section 2).

This warranty is not transferable from the original purchaser to any subsequent owner.


Important Safety Information

To ensure that you obtain the best possible results from your PxE Thermal Cycler, please carefully read the entire User Instruction Manual before use.

Once you have read and understood the contents of this manual, it is essential that the safety precautions detailed below are observed. If the PxE is not used as specified in this User Instruction Manual, the protection provided by the equipment may be impaired resulting in harm to the operator and the machine.

General Precautions• Before use, ensure that the unit has been set to the appropriate mains

voltage.• The PxE is a class 1 appliance. To minimize the risk of electric shock, the

unit must be connected to a protective earth via the supplied mains cord.• Replacement fuses must be of the correct rated current, voltage and type.• Do not operate the unit in an explosive environment.• 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 power supply and contact an authorized service center.

• 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 authorized service personnel.

• 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 surfaces without safety gloves.

• When positioning the unit, ensure that there is no restriction to the power inlet.

• Ensure that airflow to the unit is not restricted. Special care should be taken not to obstruct the vent underneath the unit, for example loose bench liner or sheets of paper can block the fan and cause damage to the unit.

• 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 Electron.

The PxE satisfies the requirements of BS EN 61010-1:1993.


Symbols and ConventionsThe PxE has been designed for safe operation. The following symbols appear on the unit and their meanings should be noted.

In addition, the following conventions are used to indicate a safety hazard

I Indicates the ON position of the main power switch.O Indicates the OFF position of the main power switch.

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: Risque de choc électrique.

GENERAL WARNING: Consult the manual for further information.ATTENTION: Lire attentivement le manuel pour plus d’information.

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.


PxE Thermal Cycler1. System Description ..................................................................9

2. Installation..............................................................................15

3. Operating Instructions............................................................17

4. Programming and Editing Methods........................................25

5. Running PCR Programs ........................................................41

6. Troubleshooting .....................................................................53

7. Maintenance ..........................................................................55

8. Technical Specifications & Ordering Information....................57

9. Appendix 1: Glossary.............................................................63

10. Appendix 2: Pre-Set Programs ............................................65

11. Appendix 3: Specifications and Ordering Information ..........67

Contents


1. System Description1.1 OverviewThe PxE is a licensed, 96 well Thermal Cycler that provides all the fundamental features required by modern researchers to perform nucleic acid amplification. The unit combines an accurate sub-ambient sample block, in 0.2ml or 0.5ml format, with on board controlling software to provide an indispensable system capable of performing both simple and sophisticated protocols.

1.2 PxE System

Power Switch

Front View

Rear View

Side View

Top View


1.3 Block FormatsThe temperature cycling blocks for the PxE system are manufactured from specially coated precision-machined aluminium, which provides excellent heat transfer from the block to the samples and also provides the additional benefit of robustness. These blocks are available in two sample formats, 0.2ml or 0.5ml and use the latest thermoelectric coupling devices (better known as peltiers) to accurately control the changes in temperature required to perform nucleic acid amplification.

The 0.2ml Sample BlockThis block allows you to use a number of consumable types from a range of manufacturers. Compatibility is assured with any of the following Thermo brand plastics:

• 1 x OmniTube 96 single piece polypropylene plate.• 96 x 0.2ml OmniStrip tubes.• 96 x 0.2ml individual OmniTubes.

The 0.5ml Sample BlockThis block has the capacity to accommodate up to 48 standard 0.5ml individual OmniTubes. In similar fashion to the 0.2ml block, the design will accept 0.5ml tubes from a range of manufacturers.

1.4 Hotlid DesignThe Hotlid enables the running of temperature cycling protocols without the need for paraffin or mineral oil overlays. Such vapor barriers have been required in the past to prevent evaporation before thermal cycling is complete. The Hotlid 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, minimizes 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.

Operation of the Hotlid on the PxE is extremely easy. An upper or lower setting can be selected depending on your pressure requirements and consumable used.


Open Lid Icon Upper Setting Icon Lower Setting Icon

WARNINGWARNING!BURN HAZARD!The tops of reaction vessels and the surfaces of the Hotlid (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.


1.5 User Interface

Operating KeysKEY FUNCTION

MENU KEYWhen this key is pressed, the MAIN MENU is displayed – (can be used as an “escape” key).

ENTER KEY Used to select highlighted Menu and program choices.

4-WAY DIRECTIONAL ARROWS

The cursor displays on the screen as a dark flashing square. The directional arrow key can be used to move the cursor between various options which are then selected by depressing the ENTER key.

ALPHA NUMERIC KEYS

When entering parameters such as program names and selecting programs, the alphanumeric keypad should be used.

STOP KEYCancels instructions / operations / programs and can be used to delete programs and directories.

BLOCK INFORMATION KEY

Provides access to current / historical information on block status screens.

PAUSE KEY Pauses a cycling program at current step.

CONTINUE KEYUsed to progress from “Hold”, “Pause” or “Self-Check” instructions. Also used to access advanced edit functions.

Pause

Block

Stop

Continue


1.6 Accurate Temperature ControlAccurate temperature control is a primary requirement for a modern thermal cycler. The way in which heat is transferred from the peltier devices to the reaction mix can have a large impact on the success and quality of your PCR result. Subsequently, the PxE has been designed with this in mind and provides multiple temperature control options to ensure consistently high performance across a range of protocols.

1.7 Simulated Tube / Plate ControlSimulated Tube Control or Simulated Plate Control are the most sophisticated temperature control options available on the PxE and are the method of choice for a number of PCR applications. These methods of temperature control work by heating (or cooling) the block in accordance with the set temperature from your protocol. While the block is heating (or cooling), measurements are taken of the temperature achieved and an algorithm is applied from the software to control whether more heating (or cooling) is required.

Using this method accounts for the lag in the transfer of the heat from the peltier, through the block and into the reaction mix. Furthermore, as their names describe, these methods account for whether you are using plates or tubes, an important factor when you consider that these consumable types can have different thermal transfer characteristics.

1.8 Block ControlBlock control is the most basic type of temperature control available and is usually only recommended for protocols that consist of long static incubations. This method does not account for the lag in temperature transfer between the peltiers and the samples and therefore relies simply on adjusting the temperature in accordance with a measurement of the block temperature.

1.9 UniformityEvery machine manufactured by Thermo Electron Corporation is calibrated using miniature thermistor probes located in tubes. These are placed at several block positions simultaneously, ensuring the required temperatures and incubation times achieved are uniform in all samples.


1.10 Specifications

Block formats0.5ml standard0.2ml standard

Temperature control availableSimulated plate controlSimulated tube control

Block controlBlock temperature range 4° C - 99° CBlock heating rate Up to 3° C / secBlock cooling rate Up to 2° C / secBlock uniformity ± 0.5° C within 30 secondsDisplay resolution 0.1°CNumber of protocols 99Number of directories 6 (5 Editable)Maximum number of protocol stages 10Maximum number of steps per stage 10Pause facility YesAdjustable temperature ramping YesTime increment / decrement YesTemperature increment / decrement YesHold function YesAuto restart facility following power failure YesRun ‘end time’ calculation YesProgram naming YesAbility to edit during cycling YesEnd run alarm YesCopy protocols Yes


2. Installation2.1 UnpackingWARNINGWARNING!

Before unpacking the PxE, please make sure that the outer packaging is undamaged.

Packing List • PxE Unit• PxE User Instruction Manual• Power Cord

Please check you have the correct block type by referring to the logo on the front of the machine. If any item is missing or damaged, please contact Thermo Electron or your local authorized distributor.

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 Electron Corporation cannot accept responsibility for any damage incurred if the unit is incorrectly packed and transported.

2.2 Setting Up Your PxEChoosing A LocationTo ensure the best operation and longevity from your PxE, please consider the following when choosing a location:

• Place the unit on a stable and level surface.• Connect to a mains supply free from voltage fluctuation.• Accommodate airflow requirements.• Ensure easy access, placing the unit under shelves may restrict access

and airflow.• Ensure that the environment is non-explosive.• Avoid areas of excessive dampness.

Environmental RequirementsThe PxE has been tested to ensure that it meets its published specifications when operated indoors under the following conditions:

• Ambient temperatures between 4°C and 35°C.• Relative humidity up to 80%.• Altitudes between 0 and 2000m.

WARNINGWARNING!If the unit is transported or stored in conditions of high humidity it must be allowed to stabilize at normal ambient temperature before powering up the unit.


2.3 Power Supply Requirements550W at 115/230V a.c. ±10% and 50/60Hz.

The PxE has a power input socket located at the back of the unit. Where possible, avoid connecting the PxE to a power supply subject to voltage fluctuations, e.g. a circuit shared by an ultracentrifuge or refrigerator.

2.4 Airflow RequirementsIn the PxE, excess heat is removed from the system by a fan which requires uninterrupted airflow for normal operation. The air intake for the fan is located underneath the unit and air is expelled up through the lid of the unit.

To ensure correct airflow around the unit, allow at least 8cm clearance between the sides of each module.

WARNINGWARNING!Special care should be taken not to obstruct the vent underneath the unit with items such as loose bench liner or sheets of paper. The unit must never be covered during operation.Even with the protective block surround, it is preferable to keep the unit away from strong currents of hot or cold air. This will ensure that there are no ‘edge effects’ where PCR performance is diminished around the exterior wells due to fluctuations from the set temperature as a result of drafts.

2.5 Use in Cold roomsThe PxE can be successfully operated in a cold room environment at 4° C. When the PxE thermal cycler is removed from a cold room, it should be left to achieve equilibrium prior to further use for 2-3 hours to avoid condensation.


3. Operating Instructions3.1 Initial Start UpPower UpThe power cord should be attached at the rear of the unit. Switch on the PxE using the power switch adjacent to the power input jack.

Self-Check ProcedureTo ensure consistent performance of the PxE, when the unit is first switched on it will complete a start up and self-check routine. After a few seconds the screen will display SELF-CHECK PROCEDURE IN PROGRESS and a percentage value to indicate the proportion of the self-check that has been completed. In addition, the cooling fan will power up.

The self-check routine ensures the block thermistor, heating and cooling circuits and the heated lid are all functioning properly. This routine takes approximately 30 seconds.

WARNINGWARNING!As the block will become hot during the self-check procedure, please keep the lid closed.

I ON position

O OFF position

S E L F - C H E C K P R O C E D U R E

I N P R O G R E S S

X X % C O M P L E T E D


3.2 Main Menu OptionsAfter successful start up, the MAIN MENU is displayed. It is possible to return to this menu at any time by pressing ‘MENU’. The Main Menu displays the date, time and the status of the block (active, inactive, or active resumed).

All aspects of the programming are accessed through the Main Menu.

Table 3.1 Summary Of Main Menu Functions

3.3 Set Up FunctionsFrom the MAIN MENU, use the directional arrows to position the flashing cursor over SETUP. Press ENTER to access the SETUP menu to allow programming of custom parameters.

The SETUP menu also displays the current software version and block type.

ClockThe time and date format of the clock display can be modified. To set the clock or alter the format, position the flashing cursor over CLOCK from the SETUP menu and press ENTER.

The American (MM/DD/YY) or European (DD/MM/YY) format can be selected using the arrow keys.

Press ENTER or the down arrow to move to the time / date setting fields. The alpha numeric keypad can now be used to enter Hours (HH), Minutes (MM), Day (DD), Month (MM), Year (YY) information. Press ENTER to return to the Main Menu.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U

R U N M A N P R O G S E T U P

RUN Select RUN to run an existing program.MAN Select MAN to run a manual static incubation.PROG Select PROG to edit, copy, view or erase a program.

SETUPSelect SETUP to alter the time, date, and power resume function and display contrast settings and to identify software version fitted.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

B L K : 0 2 V 0 0 . 0 1

S E T U P M E N U

C L O C K P O W E R D I S P L A Y

T I M E A N D D A T E

F O R M A T D D M M Y Y

H H M M D D M M Y Y

1 2 0 1 0 1 0 1 0 3


3.4 Power Failure OptionsIf there is a mains power interruption during a run, the PxE can be programmed to either automatically resume, or abandon the program that was running once power is restored. The instrument will record the duration of the interruption and the step, stage and cycle number at which it occurred. Please note: if there have been several breaks in the power supply, only the most recent interruption is recorded.

Select POWER from the SETUP menu and press ENTER. The ABANDON or RESUME options can be selected by pressing the or keys. Press ENTER to return to the Main Menu.

3.5 Display AdjustmentThe display brightness can be adjusted for varying light conditions.

Select DISPLAY from the SETUP menu, and press ENTER. Use the or keys to increase or decrease the screen brightness respectively. Press ENTER to save the current screen brightness and return to the Main Menu.

3.6 Run FunctionLoading Your SamplesThe PxE will produce identical cycling profiles whether 1 or 96 samples are to be analyzed. The following guidelines maximize the uniformity and thermal transfer characteristics of the block:

• Load samples uniformly across the block rather than in clusters. Spreading the thermal load in this way maintains uniformity.

• If small numbers of samples are to be run, please use dummy tubes to ensure that there is at least one tube in each quadrant of the block. This facilitates even heated lid pressure for good thermal transfer to the tubes.

• Care should be taken when matching block, consumable, and control mode and sample number (as shown below).

• Ensure tube caps are properly closed before loading the block.

P R O G R A M R E S U M E A T

E N D O F P O W E R F A I L U R E

< E N T E R > T O C O N T I N U E

A D J U S T D I S P L A Y

L I G H T

D A R K



Table 3.2 Consumable Compatibility Table

TypeRecommended Control Method*

Block0.5ml

Block0.2ml

Notes

0.2ml Tubes SIM TUBE NO YES

Maximum of 96 tubes / block. Tubes should be spaced out evenly to maintain uniformity. Suitable for use with flat caps or domed caps.

0.2ml Strips SIM TUBE NOYES 12 strips of 8 / block. Suitable for

use with flat caps or domed caps.

OmniTube 96 Standard Profile Polypropylene Plate

SIM PLATE NO YES

Plate can be used with a number of sealing options including domed and flat caps, adhesive and heat seals or OmniSeal Mat. Certain consumables require the use of a compression pad.

OmniFast 96 Low Profile Polypropylene Plate

SIM PLATE NO YES

Plate can be used with a number of sealing options including domed and flat caps, adhesive and heat seals or OmniSeal Mat. Certain consumables require the use of a compression pad.

0.5ml Tubes SIM TUBE YES NO

Maximum of 48 tubes / block arranged in a diagonal pattern e.g. A2, B1, A4, B3, C2, D1 etc. Tubes should be spaced out evenly to maintain uniformity. Suitable for use with flat caps or domed caps.

0.3ml Tubes SIM TUBE YES NO

Maximum of 96 tubes / block. Tubes should be spaced out evenly to maintain uniformity. Suitable for use with flat caps or domed caps.

Racked OmniTube 96 Standard Profile Polypropylene Plate

SIM PLATE YES NO Seal using flat or domed strip caps.

* See Section 5 for further details


3.7 Hotlid OperationThe adjustable Hotlid enables the running of temperature cycling protocols without the need for paraffin or mineral oil overlays. Such vapor barriers have historically been required to prevent evaporation before thermal cycling is complete. Operation of the hotlid on the PxE is extremely easy. It works by positioning a heated plate in contact with the top of the reaction vessel. This heated plate then heats the air in the dead space above the reaction mixture to a temperature that is permanently higher than the sample temperature. This elevated air temperature, relative to the sample temperature, minimises evaporation of the reaction mixture as it is repeatedly heated and cooled.

The Hotlid has two simple settings, upper or lower that can be selected depending on your pressure requirements and the consumable used.

Opening and Closing the LidTo open the lid, rotate the knob counter clockwise so that the open lid icon (see Figure 3.1) is aligned with the front of the unit. This releases the catches at the front of the block, allows the gas strut and hinges to raise the lid and expose the block recess.

To close, gently lower the lid until the catches engage and there is an audible click.

Setting The Correct Height of the Heater Plate to Suit Consumable TypeThe action of closing the lid automatically positions the heater plate to the default upper setting, which should be used in conjunction with tubes and plates of standard profile.

• If using low profile plates, the height of the lid can be adjusted to the lower setting by rotating the knob clockwise so that the relevant icon is aligned with the front of the unit (see Figure 3.1).

Open Lid Icon Upper Setting Icon Lower Setting Icon

Figure 3.1 Hotlid Icons In Detail

The temperature of the heating plate is set at 120°C (max. surface temperature) with power control affected 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 minutes before cycling commences. The block can be operated with or without the lid being switched on.


WARNINGWARNING!BURN HAZARD!Both the tops of reaction vessels and the surfaces of the Hotlid 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.

3.8 Running A ProgramTo allow immediate use of the PxE, the unit is delivered with 10 pre-stored programs. Running one of programs is easy.

At the Main Menu, with the cursor on RUN, press ENTER

Move the flashing cursor to the appropriate source directory (e.g. F:THERMO) using the or keys and press ENTER.

Use the or keys to scroll through the stored programs (for a full details of the stored programs see Appendix 3). Select program number to be run by pressing ENTER.

Once the program has been selected, user defined program parameters can be selected by following the on screen prompts.

Prior to starting the program pressing the MENU key at any stage will return to the Main Menu, aborting the run. More details can be found in Section 5.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


S E L E C T D I R E C T O R Y

A : D :

B : E :

C : F : T H E R M O

R U N F : 9 0 3 T P C R

H O T L I D A U T O

L O A D I N G A L A R M O F F

E N D R U N A L A R M O F F


3.9 Manual FunctionThis function is used for static incubations when thermal cycling is not required, e.g. hot start protocols, probe denaturations, enzyme reactions. In this mode the unit will operate under block control (see Section 5 for details).

To run a manual incubation, form the MAIN MENU use the arrow keys move the flashing cursor to MAN and press ENTER.

Use the alphanumeric keypad to enter the name of the user (or protocol) if desired. Use the or keys to scroll through the alphabet and the or

arrows to move to the next / previous character. Press ENTER to save changes and move on to the next option.

Set the desired temperature using the alphanumeric keys. Press ENTER to continue.

Use the or arrows select the heated lid on (auto) or off and press ENTER to start the incubation.

The screen displays the set temperature and actual temperature measured by the block thermistor, together with the elapsed time. The count-up timer will start when the block reaches the desired temperature.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


M A N U A L U S E R X X X X X X X

T E M P

H O T L I D O F F


T E M P 4 5 . 0

H O T L I D O F F


T E M P 4 5 . 0 T I M E 0 0 : 0 0

B L O C K 4 5 . 0

< C O N T I N U E > N E W T E M P


Please Note: If the machine is started from ‘cold’, approximately two minutes will be required before the Heated Lid is at operating temperature (based on 20°C ambient and 230V supply. This may vary under different power conditions). Condensation may be experienced if samples are loaded before the lid is at operating temperature.

Press to change the set temperature.

Press twice to cancel the manual program.

Programming FunctionThe PxE provides a range of easy options for programming and editing PCR protocols.

Use the or arrows to move the flashing cursor to PROG in the MAIN MENU.

This displays the PROGRAM MENU, which allows access to the four main editing functions.

• EDIT: To alter the details of an EXISTING program or to create a completely NEW program. Please note that if NEW is used, it will automatically move to the next available program number.

• COPY: Use the screen prompts to copy existing programs stored on the PxE from one directory or program location to another.

• VIEW: To check the details of an existing stored program.• ERASE: To erase the details of a program, setting the parameters to zero.

If at any time you wish to stop programming, press MENU to return to the Main Menu and abort the current action.

Program editing is discussed in detail in Section 4.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

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0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

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E D I T C O P Y V I E W E R A S E


4. Programming and Editing Methods

4.1 IntroductionProgramsThe PxE is designed to make programming easy. Thermal cycling protocols are stored in the instrument memory as programs. The memory will hold up to 99 complete thermal cycling protocols including 10 pre-programmed template programs (Nos. 90-99 detailed in Appendix 3). Programs are identified by a number (01-99) and a user defined name (up to 7 characters).

Programs can be manipulated in a number of ways. All aspects of program editing, such as writing new and editing existing programs are accessed though the PROGRAM MENU.

DirectoriesOnce a program has been written, it can be stored in one of five directories (A-E), which can be individually named. The sixth directory, F: THERMO is reserved for pre-set programs. When a new program is written, it can be stored in a directory of choice. This allows a series of programs to be grouped together by a name, which could define a particular user, project or lab.

Directories can also be erased or renamed as required.

Once assigned to a directory, a program can be copied to another program space in either the same or a different directory.

4.2 Stages and Steps of a ProgramTo make programming easy, the programs in the PxE are divided into a series of ‘stages’ and ‘steps’, which are displayed on the screen. In a simple PCR program, a stage typically represents a major part of a protocol, such as an initial denaturation, the cycling parameters or the final extension. The steps make up the smaller components of these stages and typically include parameters for the denaturation, annealing and extension (see Table 4.1 below).

Table 4.1 Typical Three Temperature PCR Protocol

Stage Number Step Number Temperature Dwell TimeNumber of Cycles

Stage 1 Step 1 94°C 0:01:00 x 1

Stage 2Step 1 94°C 0:00:30 )Step 2 55°C 0:00:30 > x 30Step 3 72°C 0:00:30 )

Stage 3Step 1 72°C 0:05:00 x 1Hold 4°C


The PxE allows you to program up to 10 separate stages each containing a maximum of 10 separate steps. In addition each stage can be repeated for up to 99 cycles, and a ‘hold’ added to the end of the stage. This means that even the most complex thermal cycling protocol may be saved in a single program space.

4.3 Creating A New DirectoryFrom the MAIN MENU, use the or keys to move the cursor to PROG, press ENTER to select.

The PROGRAM MENU is now displayed. With the cursor over EDIT press ENTER

The PROGRAM EDITING MENU is now displayed. Use the or keys to select NEW and press ENTER.

The NEW MENU is now displayed. Use the or keys to select NEW DIR and press ENTER.

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0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

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E X I S T I N G N E W

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

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N E W M E N U

N E W P R O G N E W D I R


Use the or keys to navigate the cursor to the desired directory (A-E). Press ENTER. You are now able to name the directory (maximum of seven characters), using the or keys to scroll through the characters or the alphanumeric keypad. Move to the next / previous character using the t and u keys.

Press ENTER only when the name is complete to save changes and return to the MAIN MENU. If no characters are entered, the directory name will be left blank.

4.4 Creating A New ProgramNew programs can be created in two different ways. When starting from scratch, please follow the instructions below. Alternatively, it is possible to copy an existing program and modify using the EXISTING option in the PROGRAM EDITING MENU.

From the MAIN MENU, use the or keys to move the cursor to PROG, press ENTER to select.

The PROGRAM MENU is now displayed. With the cursor over EDIT press ENTER.

The PROGRAM EDITING MENU is now displayed. Use the or keys to select NEW and press ENTER.

E N T E R N E W N A M E

A : X X X X X X X

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0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

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The NEW MENU is now displayed. Select NEW PROG by pressing ENTER.

Use the or keys to move the cursor to desired target directory and press ENTER to select.

The instrument software automatically allocates the next available program space. Enter the name of the protocol (maximum of seven characters) using the or keys to scroll through the characters or the alphanumeric keypad. Move to the next / previous character using the or keys.

Press ENTER only when the name is complete to save changes. If no characters are entered, the default NO NAME will appear. Please note: If there are no free program spaces the PxE will prompt you to overwrite an existing program.

The protocol parameters can now be entered. Use the alphanumeric keypad to enter the relevant values for temperature and time (hr:min:sec).

Use the or keys to move across menu. Use ENTER or to advance to the next step of the protocol. If a value is entered incorrectly, the or

keys can be used to scroll backward and forward to edit the mistake. The program will not allow you to advance to step 2 until a valid temperature and time have been entered.

Continue to enter the remaining times and temperatures of this stage. If none are required at the stage of the protocol, press ENTER (leaving each parameter at 0:00) until you access the number of cycles screen. Enter the number cycles for this stage using the alphanumeric keys. A hold step can also be programmed after each stage. This is particularly useful for ‘hot start’ protocols. If a hold step is not required, press ENTER to reach the next stage.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

N E W M E N U

N E W P R O G N E W D I R


A : D :

B : E :

C : F : T H E R M O

N E W A : 0 1 X X X X X X X


S T A G E 0 1 S T E P 0 1

T E M P 9 5 . 0

T I M E 0 : 0 1 : 0 0


Repeat the process above to enter any additional stages and steps that are required (up to a maximum of 10). Press ENTER (or key) with each parameter at zero to finish the last stage of your protocol.

Enter a hold temperature if required. If a hold temperature is not entered following the last step of the protocol, the instrument will cool to room temperature once the protocol is completed. Press ENTER to access the SAVE screen.

Press ENTER to save changes and return to the MAIN MENU.

4.5 Editing An Existing ProgramWhen editing a program and saving as a different program, it is recommended that the COPY function should first be used to save the program to a different program space (see below) and then the copy should be edited.

The program name can be edited or deleted at this stage although it will be saved unchanged if the enter key is pressed without changing the data.

It is possible to edit programs whilst the PxE is running a different protocol.




S T A G E N U M B E R 0 1

N U M B E R O F C Y C L E S 0 1

H O L D T E M P 0 0 . 0

S A V E A : 0 1 X X X X X X X

< E N T E R > T O S A V E

< M E N U > T O R E T A I N

O L D P R O G R A M

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E




The PROGRAM EDITING MENU is now displayed. Use the or keys to select EXISTING and press ENTER.

It is now possible to select the directory (A-E) where the program to be edited is stored. Use p or q to move the cursor to desired directory and press ENTER to select. Please note: The programs stored in the F:THERMO directory cannot be edited.

Use the or keys to scroll through the programs stored in the selected directory. When the name of the program to be edited appears, press ENTER to continue. If required, the program name can be edited. Usethe or keys to scroll through the characters or the alphanumeric keypad. Move to the next / previous character using the or keys. Press ENTER to accept changes and continue on to protocols parameters.

Use tthe or keys move across the menu, and or ENTER to advance through the fields. Time and temperature parameters can be altered using the alphanumeric keys as required.

To increase the number of steps in a stage, simply input values into the zero time / temperature fields. To decrease the number of steps in a stage just input the new data over existing time / temperature values.

To complete editing stage 1, press ENTER with each parameter set at zero. Enter any changed cycle number or hold temperature values. Press ENTER to proceed to the next stage. Please note: If only minor modifications are required, once the changes have been made, press the MENU key followed by ENTER to save changes and return to the Main Menu. Alternatively, proceed to remaining stages as required.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E




A : D :

B : E :

C : F : T H E R M O

E D I T A : 0 1 X X X X X X X


E D I T I N G A : 0 1 X X X X X X X

S T A G E 0 1 S T E P 0 2

T E M P 9 4 . 0

T I M E 0 : 0 1 : 0 0


Edit steps in further stages as required. Press ENTER with each parameter at zero to access the save screen.

Press ENTER to save changes and return to the MAIN MENU.

4.6 Advanced Program EditingThe Advanced Edit features allow the creation of more complex cycling protocols in order to enhance experimental data. It is possible to program increments or decrements of both time and temperature on a cycle-by-cycle basis. In addition, the rate at which the temperature changes between two given temperatures (ramp rate) can also be user defined.

Pressing the key at the temperature step at which the function is to take effect accesses advanced Edit features. If Advanced Edit data already exists for any step - <ADV> is indicated on the screen.

4.7 Programming Time / Temperature Increment / Decrements

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 to perform Touchdown PCR.

If Advanced Edit parameters are to be added to an Existing program, the Advanced Edit feature can also be accessed from the normal PROGRAM EDIT MENU. If desired, the Advanced Edit feature can be deleted for the current step. To do this, set all the parameters back to zero. The <ADV> message will then not be shown in the temperature / time set up screen, denoting that no Advanced Edit functions are operating.

4.8 Time Advanced Edit Worked ExampleBelow is an example of a protocol where the extension step is fixed for the first 15 cycles of a 25-cycle program and increased by 5 seconds/cycle for the next 10 cycles, to compensate for loss of enzyme activity:

S A V E A : 0 1 X X X X X X X

< E N T E R > T O S A V E

< M E N U > T O R E T A I N

O L D P R O G R A M



Stage 2

Step 1 95°C 0:00:30 )Step 2 55°C 0:00:30 > x 30

Step 3 72°C 0:00:30 + 5s/cycle

)


To enter this program, use EDIT functions (NEW or EXISTING) as described above to navigate to Stage 2, Step 3.

Press the key to access the Advanced Edit Screen.

Use the or keys or ENTER to move through the fields until you reach the desired parameter. Use the or keys to toggle between Increment and Decrement in set point time / cycle. Use the alphanumeric keypad to enter the amount by which the set point time per cycle should change (min:sec) using the number keys. (E.g. an increase of 5 sec per cycle). Press ENTER to accept changes and return to relevant stage and step of protocol. <ADV> now appears on the screen indicating an Advanced Edit function has been programmed.

It is possible to alter both the RAMP speed and TEMP increment / decrement parameters in this screen using the same keys. These altered parameters would also be executed, simultaneously during your experiment.

E D I T I N G A : 0 1 T I M E I N C

S T A G E 0 2 S T E P 0 3

T E M P 7 2 . 0

T I M E 0 : 0 0 : 3 0

S T A G E 0 2 S T E P 0 3

R A M P 0 . 0 0

T E M P I N C 0 . 0 0

T I M E I N C 0 : 0 0

E D I T I N G A : 0 1 T I M E I N C

S T A G E 0 2 S T E P 0 3

T E M P 7 2 . 0

T I M E 0 : 0 0 : 3 0 < A D V >


4.9 Temperature Advanced Edit Worked ExampleBelow is an example of a protocol where the annealing step is fixed for the first 5 cycles of a 25 cycle program and decreases by 1.0°C/cycle for the next 15 cycles, to reduce specificity and increase yield as product accumulates (a “touchdown” protocol).

To enter this program, use EDIT functions (NEW or EXISTING) as described above to navigate to Stage 2, Step 2.


Usethe or keys or ENTER to move through the fields until you reach the desired parameter. Use the or keys to toggle between Increment and Decrement in set point temp / cycle. Use the alphanumeric keypad to enter the amount by which the set point temp per cycle should change (°C). (E.g. 1.0°C decrease per cycle).

Press ENTER to accept changes and return to relevant stage and step of protocol. <ADV> now appears on the screen indicating an Advanced Edit function has been programmed.



Stage 2

Step 1 95°C 0:00:30 )

Step 2 65°C 0:00:30 – 1.0°C/cycle

> x 16

Step 3 72°C 0:00:30 )


E D I T I N G A : 0 2 T E M P D E C

S T A G E 0 2 S T E P 0 2

T E M P 6 5 . 0

T I M E 0 : 0 0 : 3 0

S T A G E 0 2 S T E P 0 2

R A M P 0 . 0 0

T E M P D E C 1 . 0 0

T I M E I N C 0 : 0 0

E D I T I N G A : 0 2 T E M P D E C

S T A G E 0 2 S T E P 0 2

T E M P 6 5 . 0

T I M E 0 : 0 0 : 3 0 < A D V >


4.10 Changing Temperature Ramp RatesThe 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 stabilize the primer/template duplex.

Ramp rates can be altered in the Advanced Edit screen. The default ramp rate (0.00oC / sec) is “as fast as possible”. The settable range is 0.01oC / sec to 9.99oC / sec, with the maximum practical setting being 3.00oC / sec with current technologies. The data entered refers to the ramp rate to the CURRENT step from the PREVIOUS temperature set point.

To change the ramp rate from the default setting, use EDIT functions (NEW or EXISTING) as described above to navigate to the desired step, for example Stage 2, Step 2.


Use the alphanumeric keys to change the current ramp rate setting. Press ENTER to accept changes and return to relevant stage and step of protocol. <ADV> now appears on the screen indicating an Advanced Edit function has been programmed.

E D I T I N G A : 0 3 R A M P

S T A G E 0 2 S T E P 0 2

T E M P 5 5 . 0

T I M E 0 : 0 0 : 3 0

S T A G E 0 2 S T E P 0 2

R A M P 0 . 0 0

T E M P I N C 0 . 0 0

T I M E I N C 0 : 0 0

E D I T I N G A : 0 3 R A M P

S T A G E 0 2 S T E P 0 2

T E M P 5 5 . 0

T I M E 0 : 0 0 : 3 0 < A D V >


4.11 Copying ProgramsCopying programs is necessary in, for example, modifying the pre-set programs described in Appendix 3.


The PROGRAM MENU is now displayed. With the cursor over COPY press ENTER.

Use the or keys to navigate to the desired source directory.

The COPY PROGRAM screen is now displayed. Use the or keys to navigate to the desired source program and press ENTER.

Use the or keys to navigate to the desired target directory.

Use the or keys to select the select the desired location for the copied program. Press ENTER to copy the program and return to the MAIN MENU.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E



S O U R C E D I R E C T O R Y

A : D :

B : E :

C : F : T H E R M O

C O P Y P R O G R A M

F R O M F : 9 0 3 T P C R

T A R G E T D I R E C T O R Y

A : D :

B : E :

C : F : T H E R M O

C O P Y P R O G R A M

F R O M F : 9 0 3 T P C R

T O A : 0 4 E M P T Y

< E N T E R > T O C O P Y


Note that if a location with an existing program is selected as the target program, any data in the target program space will be overwritten.

4.12 Viewing An Existing ProgramIt is possible to view existing programs without altering the information contained within them.


The PROGRAM MENU is now displayed. With the cursor over VIEW press ENTER.

Use the or keys to move the cursor to the relevant source directory and press ENTER.

Use the or keys to scroll through the available programs in the source directory. When the relevant program appears on the display, press ENTER to view.

A L L 0 4 E M P T Y D A T A

W I L L B E O V E R W R I T T E N

< E N T E R > T O C O P Y

< M E N U > T O A B O R T

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E




A : D :

B : E :

C : F : T H E R M O

V I E W A : 0 4 3 T P C R


< M E N U > T O S T O P


Use the ENTER key and the or keys to move through the program, and CONTINUE to enter and leave the Advanced Edit information.

The MENU key closes the view screen, and returns to the Main Menu.

4.13 Erasing A DirectoryDIRECTORIES CAN BE ERASED TO VACATE PROGRAM SPACE FOR FUTURE USE.From the MAIN MENU, use the or keys to move the cursor to PROG, press ENTER to select.

The PROGRAM MENU is now displayed. With the cursor over ERASE press ENTER.

The ERASE MENU is now displayed. With the cursor over ERASE DIR press ENTER.

Select the directory be erased.

V I E W I N G A : 0 4 3 T P C R

S T A G E 0 3 S T E P 0 1

T E M P 7 2 . 0

T I M E 0 : 0 5 : 0 0

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E



0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

E R A S E M E N U

E R A S E P R O G E R A S E D I R


A : D :

B : E :

C : F : T H E R M O


Confirm erasure of the program by pressing STOP ( ) as prompted or MENU to keep the directory.

4.14 Erasing A ProgramPrograms can be erased to vacate program space for future use.


The PROGRAM MENU is now displayed. With the cursor over ERASE press ENTER.

The ERASE MENU is now displayed. With the cursor over ERASE PROG press ENTER.

Select the directory containing the program to be erased.

A L L P R O G R A M S W I L L B E

E R A S E D I N A : N O N A M E

< S T O P > E R A S E P R O G R A M

< M E N U > K E E P P R O G R A M

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E



0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

E R A S E M E N U

E R A S E P R O G E R A S E D I R


A : D :

B : E :

C : F : T H E R M O


Use the or keys to select the program to be erased and press ENTER.

Confirm erasure of the program by pressing STOP ( ) as prompted or MENU to keep the program.

4.15 Editing Options While Running A ProtocolTo ensure that the time the PxE is running programs is maximized, it is possible to access all editing options while running a program. If the RUN screen is displayed (see chapter 5 for full details), press MENU to return to the Main Menu where the block status is defined as ACTIVE.



E R A S E P R O G R A M

A : 0 4 3 T P C R

< E N T E R > T O E R A S E

E R A S I N G A : 0 4 3 T P C R

< S T O P > E R A S E P R O G R A M

< M E N U > K E E P P R O G R A M

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

A C T I V E

M A I N M E N U


0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

A C T I V E




5. Running PCR Programs5.1 The Run FunctionThe RUN Function in the MAIN MENU enables users to start a previously stored program. If the program you would like to run has not been created, see Section 4 for instructions on how to create a new program.

To allow immediate use of the PxE, the instrument is supplied with ten (non-editable) pre-set protocols, stored as programs 90-99 in the F:THERMO directory. These pre-set protocols cover the most common thermal cycling techniques, and can be used to create customized protocols by editing (for full protocol details see Appendix 3).

When running a program on the PxE, several user defined parameters need to be considered prior to starting the program. These include the following:

• Temperature control method to be used.• Operating mode of heated lid.• Use of alarms to indicate loading and program completion.


5.2 Temperature Control OptionsCrucial to the accurate operation of a thermal cycler is an understanding of the temperature control methodology. Careful consideration should be made relating to which type of temperature control method is used because this parameter impacts heavily on PCR performance and experimental run times. The PxE can control the temperature in two main ways: Simulated Control and Block Control.

Simulated ControlThis temperature control method uses an algorithm to account for the rate of thermal transfer from the block to the reaction mix. Due to the inherent thermal load of the samples, there is a difference in the temperature achieved by the block and the temperature of the samples positioned in the block. When heating and cooling samples with Simulated Control, the software monitors the block temperature and uses the calculated values of the software algorithms to provide a determined overshoot (or undershoot). This means that the lag in thermal transfer to the samples is eliminated and the temperatures achieved more accurately represent those that were set in the program. (See Figure 5.1).

Figure 5.1: Simulated Temperature Control (Tube or Plate)

For optimal PCR performance on the PxE, two methods of simulated control are available for use on the PxE, one for protocols performed with tubes and the second for those that use plates. These methods use different algorithms to account for differences in heat transfer due to the consumable type.


5.3 Simulated Tube Control (SIM TUBE)Reactions using Simulated Tube Control require number of samples and volume details to be entered when prompted by the run screen. For all tube reactions in a 0.2ml or a 0.5ml thin walled tube, the sample volume is the total reaction volume in µl in one well (including any oil overlay). For reactions in thick walled 0.5ml tubes, use (volume + 50) µl as a good approximation to achieving the same profile.

5.4 Simulated Plate Control (SIM PLATE)This mode of control operates on a similar principle to simulated tube control. However in this case, the control algorithm has been adjusted so that the programmed temperature is achieved in the volume inside a plate well.

The use of 96 well plates on the 0.5ml block should be treated with caution. 96 well PCR plates have been used successfully on the 0.5ml block. However, the fit is not optimal, and as a result there is a significant air gap between the plate and the block itself and the volume factor should be used to compensate. We recommend initially implementing a sample volume factor equal to 10 x reaction volume, but further optimization may be necessary.

5.5 Block ControlBlock control provides less accurate control of sample temperature and does not take into account the differences in thermal transfer from the block to the reaction mix seen when using different sample volumes and consumable types. For this reason, Block control is only recommended for prolonged static incubation periods. Please Note: Due to the lag in thermal transfer, the reaction mix is incubated at the set temperature for a considerably shorter time than programmed.

Figure 5.2: Block Control


5.6 Operating Methods For The HotlidHotlid AutoWhen selected in Auto mode, the Hotlid undergoes a preheat phase. Once completed and the lid is at operating temperature, the program selected to run will automatically start. This mode should be selected for robust cycling reactions because samples are loaded and the lid closed before starting the preheat i.e., where the reactants can withstand being incubated for the duration of the preheat (about 2 minutes) without the generation of spurious products.

Hotlid ManualWhen selected in Manual mode, the Hotlid undergoes a preheat phase. When the lid reaches operating temperature, an alarm will sound (if activated) and the ENTER key will need to be pressed to start temperature cycling. This should be used for sensitive cycling reactions because samples should be mixed, loaded and the lid closed after the preheat is completed i.e., where the reactants will not tolerate being incubated at low temperatures even for a short time, or where all risk of non-specific reactions must be eliminated.

If, after 90 minutes, the ENTER key has not been pressed to start a program, the lid will switch off and the block will return to the idle condition.

Hotlid OffIf the Hotlid is switched off (for example to perform experiments with oil overlay present), the temperature cycling will commence as soon as the program / control / calibration factors have been entered.


5.7 Running A ProgramAt the Main Menu, with the cursor on RUN, press ENTER.

Move the flashing cursor to the appropriate source directory (e.g. F:THERMO) using the or keys and press ENTER.

Use the or keys to scroll through the stored programs (for a full details of the stored programs see Appendix 4). Select program number to be run by pressing ENTER. Please Note: If the desired program number is known, use the alphanumeric keys to fast track to its location in the memory.

Once the program has been selected, the appropriate operating method for the Hotlid is determined. Use the or keys to toggle between available options. Press ENTER to select. If Manual Mode is selected for the Hotlid operating method, the use of a loading alarm must now be determined (under Auto mode or with the Hotlid off, the software automatically skips this option). Please Note: The or keys can be used to go backwards and forwards in the screen options at this point to correct any mistakes.

The use of an Alarm to denote the completion of the program is now determined. Use the or keys to toggle between available options. Press ENTER to select.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E

M A I N M E N U



A : D :

B : E :

C : F : T H E R M O

R U N F : 9 0 3 T P C R

H O T L I D A U T O



R U N F : 9 0 3 T P C R

H O T L I D A U T O



R U N F : 9 0 3 T P C R

H O T L I D A U T O


E N D R U N A L A R M O N


Use the or keys to toggle between available options for the Hotlid at the end of the run. Press ENTER to select.

Follow the onscreen prompts to select the desired temperature control method.

If a SIM control method is chosen the number of samples and sample volume (in µl) are now determined. Use the alphanumeric keypad to enter the appropriate values and press ENTER to confirm. If BLOCK control is chosen, this screen is skipped. Please Note: The or keys can be used to go backwards and forwards at this point to correct any mistakes.

Cycling will now commence and the run screen will be displayed if the Hotlid is selected OFF. If the Hotlid is operating under AUTO mode, the program name is now displayed and lid preheating now occurs. The heated lid typically takes around 2 minutes to reach operating temperature. During this time, the block is controlled at 4° C. Once the Hotlid has reached operating temperature cycling will commence.

WARNINGWARNING!BURN HAZARD!Both the tops of reaction vessels and the surfaces of the Hotlid 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.

R U N F : 9 0 3 T P C R

L I D O N D U R I N G F I N A L

H O L D S T E P ? N O


R U N F : 9 0 3 T P C R

S I M T U B E

P R E S S T O C H A N G E


R U N F : 9 0 3 T P C R

S I M T U B E

N O O F S A M P L E S 0 9 6

S A M P L E V O L U M E 0 2 5


If the Hotlid is operating under MANUAL start, the heated lid will preheat to operating temperature and then the unit will be paused.

Whilst the unit is paused, load samples into the block press ENTER to commence cycling.

Please note: It is possible to edit programs whilst the PXE is running a different protocol (see Section 4 for full details on editing functions).

P R O G R A M F : 9 0 3 T P C R

L I D P R E H E A T B E F O R E

A U T O S T A R T

< S T O P > T O A B O R T

P R O G R A M F : 9 0 3 T P C R

L I D P R E H E A T B E F O R E

M A N U A L S T A R T


P R O G R A M F : 9 0 3 T P C R

P A U S E D : L O A D S A M P L E S

< E N T E R > T O S T A R T



5.8 Example Run ScreensWhen a program is running three different run screens can be displayed. These contain information about the progress of the run together with the temperatures achieved during the run. The performance of the PxE can be monitored and the time of the end of the program determined.

The current block performance screen displays the following:

• Program number and name.• Current set, block and simulated temperatures.• Time remaining at current set point.

Use the or keys to access further run information.

The estimated run time screen displays the following:

• Temperature control method.• Current time.• The calculated time the program is due to complete. This time is an

estimated value that is updated throughout the run.

Use the or keys to access further run information.

The program progress and performance screen displays the following:

• The current stage and step of the running program.• The total number of cycles completed and remaining.• The maximum and minimum temperatures achieved during the run in the

respective control mode.

R U N N I N G F : 9 0 3 T P C R

T E M P 9 4 . 0

B L K 9 4 . 1 T I M E

S I M 9 4 . 0 0 0 : 2 9

R U N N I N G F : 9 0 3 T P C R

C O N T R O L S I M T U B E

T I M E N O W 1 2 : 0 0

P R O G R A M E N D 1 4 : 0 0

R U N N I N G F : 9 0 3 T P C R

S T A G E 0 2 S T E P 0 1

C Y C L E 0 5 O F 3 0

M A X 9 4 . 5 M I N 5 4 . 7


5.9 Power Failure ScreenIf there is a mains power interruption during a run, the PxE can be programmed to either automatically resume, or abandon the program that was running once power is restored (see Section 3 for full details).

Power Failure Screens When Program ResumedIn the event of a power failure with the instrument programmed to automatically resume, when the mains power is restored the instrument performs a self-check and the MAIN MENU screen is displayed.

Press the BLOCK INFORMATION KEY (i) to return to the run screens of the running program. In addition to the screens described above a Power Failure screen appears if the run has been interrupted by a power failure or power fluctuation severe enough to affect the unit. Use the or keys to view the power failure screen.

Details of the duration of the power failure and the stage in the run when the power failure occurred are displayed.

Power Failure Screens When Program AbandonedIn the event of a power failure with the instrument programmed to abandon the program, when the mains power is restored the instrument skips the self-check and the MAIN MENU screen is displayed.

Switch of the instrument and restart, allowing it to perform the self-check procedure to ensure there is no damage to the unit.

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

A C T I V E - R E S U M E D

M A I N M E N U


R E S U M E D F : 9 0 3 T P C R

O F F F O R 0 0 : 2 2 : 0 3 A T

S T A G E 0 2 S T E P 0 3

C Y C L E 1 7

0 1 / 0 1 / 0 3 1 2 : 0 1 : 0 0

I N A C T I V E - A B A N D O N E D

M A I N M E N U



5.10 End Of Run ScreensFollowing completion of the PCR Program the alarm (if selected) will sound and the block will remain at 20°C for 30 minutes to drive the Hotlid plate temperature to ambient. In addition, two end run screens are displayed. These screens provide the following information:

• The program name and number.• The maximum and minimum temperatures recorded during the run.• In the event of a mains power supply failure on a PxE that is programmed

to automatically resume, the appropriate details are displayed as described above.

• The start and finish time of the Program.

Use the key to view the start and finish time of the program.

Use the key to return to the previous screen. Press MENU to return to the MAIN MENU.

5.11 Block Idle ScreenIf the BLOCK INFORMATION KEY (i) is pressed and there is no run in progress, the block idle screen is displayed.

5.12 Error ScreensA number of error screens may be displayed if a fault has been detected in the unit. Contact the Thermo Electron Service Department or your local authorized dealer for advice before attempting to use the instrument further.

E N D E D F : 9 0 3 T P C R

M A X I M U M T E M P 9 4 . 3

M I N I M U M T E M P 5 4 . 9

E N D E D F : 9 0 3 T P C R

S T A R T E D F I N I S H E D

1 2 : 3 7 : 1 0 1 4 : 0 8 : 5 2

0 1 / 0 1 / 0 3 0 1 / 0 1 / 0 3

B L O C K I D L E

< M E N U > T O C O N T I N U E


5.13 Hold and Pause FunctionHold FunctionWhen entering a program the PxE will give you the opportunity to enter a “Hold” temperature at the end of each stage. The samples will be held at this set temperature indefinitely which can be useful in applications that require a hot start. The screen will display HOLDING during a run.

If a “Hold” is inserted between stages, press to advance the program to the next stage.

If a “Hold” is inserted at the last stage, press to switch to the run summary screen.

Pause FunctionPressing PAUSE ( ) during a cycling program will pause the program at the current or next target temperature within the step. Press to continue the countdown for the step.

5.14 Aborting ProgramsThe program will run to completion unless interrupted by the pressing of either the PAUSE ( ) or STOP ( ) keys whilst the appropriate run screen is displayed.

To ABORT a program whilst it is running, press the STOP ( ) key to display the verification screen (the program continues while this is displayed).

Press the STOP ( ) key again to abort the program. Pressing ENTER continues the program.

H O L D I N G A : 0 6 T E S T

C O M P L E T E D S T A G E 0 1

T E M P 9 5 . 0 T I M E 0 2 : 5 6

< C O N T I N U E > T O R E S U M E

P A U S E D A : 0 6 T E S T

S T A G E 0 1 C Y C L E 0 1

T E M P 9 5 . 0 T I M E 0 2 : 5 6

< C O N T I N U E > T O R E S U M E

P R O G R A M A : 0 6 T E S T




6. Troubleshooting6.1 Support ServicesThe PxE has been designed for reliability and for ease of maintenance. Thermo Electron Corporation offers full service and technical support for all its products. International Sales, Services and Technical Support contact your local Thermo Electron Corporation subsidiary or authorized distributor.

Thermo Electron CorporationSample PreparationUSA and rest of the world : +1 508 482 7000UK and Europe: +44 (0) 1256 817282

Email: [email protected]: www.thermo.com

6.2 Power Failure During a RunA message will appear on the main menu screen. The machine will either restart automatically when power is restored, or the program will be abandoned, as defined in the SET UP menu screen (see Section 3 for further details). Viewing the run screens for the block will indicate the time of the power failure, and where applicable the time the run resumed (see Section 5 for further details).

6.3 Optimization of ProtocolsThe capability of the PxE programming enables protocol optimizations to be performed very rapidly. Typically, temperature cycling protocols may consist of three distinct stages:

1. Denaturation at an elevated temperature (usually 90-95°C).2. Annealing at a temperature dictated by the melting temperature (Tm) of

the oligonucleotides.3. Enzymatic activity at a temperature dictated by the optimum temperature

of the thermostable enzyme being used.These three steps are typically repeated for 20-30 cycles depending on the amount of starting template.

DenaturationThe denaturation step at each cycle must be sufficient to denature the target DNA completely, including G-C rich regions. However, the effect on the enzyme activity of repeated high temperature incubations should also be considered. An extended initial denaturation step, (3 minutes, 95°C, before enzyme addition) will denature complex high molecular weight DNA template, but for later cycles this should be reduced to a maximum of 30 seconds at 92-95°C.

www.thermo.co


Annealing The annealing temperature depends on the size and nucleotide composition of the oligonucleotides used. In general it varies between 50°C and 70°C and as a rough guide should be 5°C below the Tm. This may be calculated approximately using the following formula:

Tm = 2 x (A + T) + 4 x (G + C)

A difference in the annealing temperature of as little as 1°C can affect the specificity of a reaction, it is therefore recommended that a range of temperatures is tested to optimize the annealing temperature for each primer and template combination.

ExtensionThe extension temperature is largely dependent upon the optimum temperature of the enzyme chosen and is usually in the range 70-75°C (see data sheet from manufacturer). The time required depends on the length of product being synthesized.

6.4 Malfunction of the UnitThe following section is a guide to isolating a particular malfunction of the unit and suggests a possible remedy to rectify the fault.Problem Possible Cause Suggested Remedy

No Power• Blown fuse.• Internal connection loose.

Isolate unit from Mains power supply. Replace fuses, if problem persists contact Thermo Electron or your authorized local dealer.

Sample Block Not Heating Or Cooling Correctly

• Displayed temperature is not rising.

• Thermo-Electric Coolers faulty or thermal fuse blown.

• Internal connection loose.

Ambient temperature could be too high, if problem persists contact Thermo Electron or your authorized local dealer.

Block Overheating

• Faulty block thermistor.• Faulty power supply.• Faulty control PCB

assembly.

Contact Thermo Electron or your authorized local dealer.

Fan fails to operate continually

• Faulty Control PCB Assembly.

• Loose fan connection• Fan unit faulty or jammed• Faulty supply connection to

Control Card

Visually check fan is free from obstruction and that there is no loose bench liner effecting airflow. If problem persists contact Thermo Electron or your authorized local dealer.

Heated Lid not heating

• Faulty connections.• Faulty lid thermistor.• Heater Faulty or thermal

fuse blown.

Check setting for Hotlid in program. If problem persists contact Thermo Electron Corporation or your authorized local dealer.

LCD ‘Blank’• Faulty connections.• Faulty LCD module.

Check that the unit is switched on. Check that the power chord is inserted correctly. If problem persists contact Thermo Electron or your authorized local dealer.

Keyboard not entering data

• Faulty connection.• Faulty Keyboard.• Faulty PCB.

Check that silicone keys are not stuck. If problem persists contact Thermo Electron or your authorized local dealer.


7. Maintenance7.1 General Cleaning All surfaces of the PxE system and Hotlid should be cleaned regularly with a soft cloth, hot water and a mild detergent.

It is important to thoroughly dry all surfaces after cleaning.

The PxE is not intended for use with aggressive chemicals and on no account should organic solvents be used to clean this equipment.

Dampened cotton buds can be used to remove dirt and debris from individual wells. The wells should be kept clean to maintain optimum heat transfer.

7.2 DecontaminationWhen 35S labelled nucleotides are thermally cycled they break down into lower molecular weight forms which are highly volatile and can leach through the walls of tubes and plates thus contaminating the block and possibly the heater plate of the Hotlid.

We therefore do not recommend the use of 35S labels, as replacing a dangerously contaminated block is expensive.

If 35S labels are used, we recommend the following to minimize contamination.

• Use a mineral oil overlay in all reactions, even when the Hotlid is used.• If using tubes, use only the thick walled variety.• Use the thermal cycler in a fume hood, to minimize air contamination.• If using plates, coat the under surface with a thin layer of mineral oil.• If radioactivity must be used, the thermal cycling block and Hotlid

surfaces can be decontaminated using a 10 % v/v solution of Neutracon (Decon Lab Ltd, Conway Street Hove, East Sussex BN3 3LY Tel: +44(0) 1273-739241, Fax: +44(0) 1273-722088) or PCC-54 (Pierce Eurochemie B.V Holland). The use of 35S labelled nucleotides is not covered under our warranty agreement and requires special service arrangements. Complete decontamination is unlikely, but low level counts can be achieved by repeated application of a fresh solution of 10% v/v Neutracon to the "Hot" area.

All Chassis, Block, and Hotlid components coming into contact with radioactivity should be decontaminated before re-use or transportation. In the event of returning a contaminated item please contact Thermo Electron Corporation’s Service Department so that the appropriate handling arrangements can be made.

Thermo Electron recommends that if radioisotopes are to be used, equipment must be located in a designated Radiation Area. Local Radiation Safety procedures must be followed at all times.


7.3 Protection to the UserThe PxE has been designed with operation safety in mind. In the rare event of an instrument failure, three levels of protection are built in to ensure the unit “fails safe”. First, the software sets normal operating ranges for the block and lid. Should this fail; electrical circuitry is in place to ensure that safe temperatures are not exceeded. In the unlikely event of this failing, thermal fuses are fitted to shut off the power supply to damaged components.

The unit is fitted with an internal lithium battery containing a hazardous substance. This should be replaced with the original type and disposed of with care.

7.4 FusesThe PxE mains power inlet is fitted with two T6.3A fuses (20mm x 5mm), which are located to the rear of the fan on the base of the unit. If necessary these may be replaced by a qualified person.

Isolate the unit from the Main Power Supply. Using a flat head screw driver simply unscrew the fuse from its housing. Discard the old fuse and replace.


8. Technical Specifications & Ordering Information

8.1 Technical Specifications

Product CodesCode Description

HBPXE02 0.2ml fixed block 96 well thermal cycler (96 well block – holds 96 tubes or 1 x 96 well plate)

HBPXE050.5ml fixed block 96 well thermal cycler (96 well block – holds 48 x 0.5ml tubes, 1 x 96 well plate)

Specification TableBlock formats Block formats 0.5ml standard 0.2ml standard

Temperature control available

Temperature control available Simulated plate controlSimulated tube controlBlock control

Block temperature range 4°C – 99°CBlock heating rate Up to 3°C / secBlock cooling rate Up to 2°C / secBlock uniformity ±0.5°C within 30 secDisplay resolution 0.1°CNumber of protocols 99Number of directories 6Maximum number of protocol stages 10Maximum number of steps per stage 10Pause facility YesAdjustable temperature ramping YesTime increment / decrement YesTemperature increment / decrement YesHold function YesAuto restart facility following power failure YesRun ‘end time’ calculation YesProgram naming YesAbility to edit during cycling YesEnd run alarm YesCopy protocols Yes


8.2 Ordering InformationMini Gel Casting Tray (60 x 90mm) HB-E4-CTNMidi Gel Casting Tray (120 x 90mm) HB-E4-CTDMaxi Gel Casting Tray (180 x 90mm) HB-E4-CTXGel Tray End Dams (set of 4) HB-E4-ED410:18 Tooth Comb (multi-pipette compatible) HB-E4-C1Prep: 14 Tooth Comb HB-E4-C2H Section for Blotting HB-E4-HSReplacement Right Electrode HB-E4-MELECRReplacement Left Electrode HB-E4-MELECLPower Cords HB-MG-PSUL8 Channel Helper HB-E4-CH

Blotting Sponges (pack of 10)Mini: HB-E4-SPMidi: HB-E4-SMMaxi: HB-E4-SX

Level Bubble HB-E4-LBLoading Mat HB-E4-LM


8.3 PlasticwareTo ensure optimal use of our thermal cyclers we recommend the use of Thermo Electron plasticware, which has been performance tested as part of our ongoing product development programme. All Thermo plasticware is manufactured under clean room conditions to ensure that the product is DNase and RNase free.

Sealing SystemsTDX Tape is single use adhesive tape sealing system for oil-free thermal cycling in 96 well plates. These tapes have been designed to prevent cross contamination between wells. After a thermal cycling reaction TDX tape can be removed from the plate with ease.

• TDX Tape is designed for use with polypropylene plates such as OmniTube 96, OmniFast 96.

TDX tape cannot be used on polycarbonate plates, as this tape will bind irreversibly to polycarbonate.

To secure contact with the heated lid of the thermal cycler the TDX tape must be used in combination with the reusable TD Compression Pad.

Consumables Pack Size Description Catalogue NumberPlates

OmniFast 96 25Polypropylene skirted 0.2ml 96 well plate

HB-TC-8002N

OmniFast 384 50 Polypropylene 384 well plate HB-TC-3840NOmniTube 96 25 Polypropylene 0.2ml 96 well plate HB-TC-6002NTubes

OmniTube 0.2ml 10000.2ml individual tubes with integral domed caps

HB-TC-3372N

OmniTube 0.2ml 10000.2ml individual tubes with integral flat caps

HB-TC-6202N

OmniTube 0.5ml 10000.5ml individual tubes with integral domed caps

HB-TC-4895N

OmniTube 0.5ml 10000.5ml individual tubes with integral flat caps

HB-TC-3505N

OmniStrip 0.2ml 2000 0.2ml strips of 8 tubes & caps HB-TC-2662NOmniStrip 0.3ml 2000 0.3ml strips of 8 tubes & caps HB-TC-4043N

OmniTube Caps 25 x 96For all 0.2ml and 0.3ml OmniTube products

HB-TC-6022N


8.4 Sealing a 96 Well Polypropylene Plate with TDX tapeWhen using any type of adhesive seal, special care should be taken to prevent creases. Position the tape over one of the short edges of the 96 well plate and peel back the lining material. Allow the adhesive layer to fall on to the plate without introducing creases. Please note: Before heating, the adhesive is soft enough to be repositioned without adverse effects.

The tape should be firmly anchored by pressing over each well. This can be done using a soft roller or by finger pressure.

Once in the machine, place the Compression Pad (HB-TD-SFOAM) on top of the sealed plate. The Compression Pad performs a dual function. It ensures that the tops of the sealed well reach the correct temperature and under compression it ensures the sealing between wells is intact.

8.5 Removing the Adhesive TapePrior to removing the TDX Tape from a plate, ensure there is no condensation of the solution on the inside the wells or on the plugs of the tape. This will minimise the risk of well-to-well contamination when removing the tape from the plate.

Tape is most easily removed while the 96 well plate is in a rack or still in the block of the thermal cycler. Peel the sheet from one corner diagonally across the plate using a single smooth action and discard.

TDX Tape is designed for single use. Once heated and compressed, their backing conforms to the plate and cannot be easily repositioned. Re-use is therefore not advised.


8.6 Sealing a 96 Well Plate Using the OmniSeal TD MatThe OmniSeal TD Mat (HB-TD-MT-SRS-5) is a re-usable silicone rubber mat designed for use with 96 well plates only (see Figure 8.1). These Mats have 96 plugs in the underside, which are spaced so that they fit into all wells of a 96 well plate and are shaped to expand widthways when pressure is applied from above by the PxE Hotlid.

Figure 8.1: Sealing of a 96 well plate using the OmniSeal TD Mat.

To minimize the risk of well-to-well contamination, remove the OmniSeal TD Mat whilst the plate is in a rack or still in the block of the machine.

8.7 Re-Using the OmniSeal TD MatThe OmniSeal TD Mat can be re-used at least 5 times. To clean and sterilize follow to brief procedure below:

• Soak for 1 minute in either 10% hypochlorite solution, or 0.1M hydrochloric acid solution.

• Rinse with distilled water.• Dry inside an oven at up to 80°C if required.• Autoclave at 121° C for 15 minutes.

Repeated washing and autoclaving of the Mats causes them to crack around the edges. When this starts to happen, they should be discarded. Please note: The number of times an OmniSeal TD Mat can be re-used is reduced considerably if it is cleaned with alcohols or detergents, or if exposed to UV irradiation.Consumables Pack Size Description Catalogue NumberSealing Systems

TDX Tape100 Individual Sheets

Sealing tape for polypropylene plates

HB-TDX-TAPE100

Compression Pad 1Foam pressure pad for use with tape sealing system

HB-TD-SFOAM

OmniSeal TD Mat 5Reusable 96 well sealing mat

HB-TD-MTSRS5


9. Appendix 1: Glossary

Advanced Edit

Allows the adjustment of the following:1) Incrementation / decrementation of time and/or temperature on a cycle-by-cycle basis.2) Control of temperature ramping between set points.

Block ControlThe temperature of the cycling block is monitored and controlled by the thermistor mounted on the block. For most cycling procedures, we recommend the use of SIM Control in preference to Block Control.

Copy Copy is used to make identical copies of stored programs, prior to editing.Edit Edit is used to create new programs or to change existing programs. Erase Erase is used to delete programs or create extra space for new protocols.

Hold

A HOLD step may be specified at the end of a stage. Samples are held at this temperature until CONTINUE is pressed to resume the program. This function can be used to hold samples at a fixed temperature while a reagent is added before temperature cycling starts.

Hotlid

A standard fitting which allows oil free cycling in 0.2, 0.3, 0.5 ml tubes and 96 well plates. The heated lid plate in contact with the top of the reaction vessels maintains the temperature above 100º C, minimizing evaporation of the sample.

MANManual operation is used for single temperature incubations under block control. The incubation will proceed at the set temperature until the STOP button is pressed, or until a new temperature is specified.

MenuThe menu key allows the user to return to the main menu at any time without making changes in the programming / operation of the unit.

New TemperatureA new temperature may be specified during a manual incubation step. The manual program is interrupted and the sample temperature rapidly changes to the newly specified temperature.

OPTS Allows the accessing of SETUP options.

Peltier Device

A thermoelectric device incorporating a bismuth telluride semi-conductor crystal array. On passing a current through the crystal, one surface of the crystal is heated, the other cooled. Reversing the current reverses the heat flow.

Power FailureMains power supply failure. The PxE can be programmed to resume or abandon the PCR run once mains supply is restored. This is specified using the SETUP menu.

PROGRAM Select PROG from the main menu to access the programming menu.Ramp Rate(Temperature Ramping)

Ramping precisely controls the rate of change of sample temperature (ºC/sec). This is useful for limiting the rate of change of temperature, to allow partial extension of short or degenerate primers.

RUN The RUN Option allows the user to run a stored program on the block.

Sample VolumeWhen using simulated tube control methods, the sample is entered to ensure precise block heating and cooling.

SET UPThis function allows the default settings of the PxE (set day and time, power restart option and screen brightness) to be modified.

Simulated Plate Control

Simulated Plate Control incorporates an algorithm to reduce time to reach target temperature, using precise overshoots in block temperature for each setpoint.

Simulated Tube Control

Simulated Tube Control incorporates an algorithm to reduce the time to reach target temperature, using precise overshoots in block temperature for each setpoint.


Stage

One stage is a major segment of a PCR program. It consists of one or more steps up to a maximum of 10. A stage may be repeated for temperature cycling. Up to ten stages may be used in one program, and stage-to-stage linking is automatic.

Step

A step consists of a programmed temperature and time interval. Time increment and ramp rate may also be specified if required. Enter zero for each heading to specify the last step of a particular stage, and enter zero for each heading at the first step of the final stage.

Sub-Ambient BlockThe aluminium block used on sub-ambient PxE systems. This uses Peltier devices for heating and cooling. Operating range: 4° C to 99° C.

TEMP INC / DECTemperature Increment / Decrement

May be used to increase / decrease a temperature set point at successive cycles. E.g. a decrement of 0.1°C will give 60, 59.9, 59.8, 59.7.... °C. This is useful for touchdown PCR reactions where stringency at the annealing step is decreased as the reaction proceeds.

TIME INC / DECTime Increment / Decrement

May be used to change a time interval to each repeat cycle of a specific step. E.g. an increment of 10 seconds on a 60 second step will give time intervals of 60, 70, 80, 90, 100....seconds. This is useful when increasing an incubation step to allow for the depletion of an enzyme.

Touchdown PCR

A gene amplification procedure where the annealing temperature is high (e.g. 65°C) to achieve high specificity at the start of the reaction. It can then be reduced with successive cycles (by e.g. 0.5° C/Cycle) to increase yield once high stringency is no longer required.

VIEW The View function allows the contents of programs to be accessed.


10.Appendix 2: Pre-Set Programs

Programs coded into the F: THERMO Directory of the PxE memory can be used directly or as building blocks for other protocols using the COPY function.

PROGRAM F:90 3T_PCRStage 1 Step 1 94°C 0:01:00 x 1

Stage 2Step 1 94°C 0:00:30Step 2 55°C 0:00:30Step 3 72°C 0:00:30 x 30

Stage 3 Step 1 72°C 0:05:00 x 1Hold 4°C

PROGRAM F:91 2T_PCRStage 1 Step 1 94°C 0:01:00 x 1

Stage 2Step 1 94°C 0:00:10Step 2 68°C 0:00:10 x 30

Stage 3 Step 1 72°C 0:05:00 x 1Hold 4°C

PROGRAM F:92 LONGPCRStage 1 Step 1 94°C 0:02:00 x 1



+ Time Inc. 0:20 / cycleStage 4 Step 1 68°C 0:07:00 x 1

PROGRAM F:93CYCSEQ

Stage 1 Step 1 94°C 0:01:00 x 1

Stage 2

Step 1 94°C 0:00:10Step 2 50°C 0:00:10Step 3 60°C 0:04:00 x 25Hold 10°C

PROGRAM F:94 TD_PCRStage 1 Step 1 94°C 0:01:00 x 1



Stage 3

Step 1 94°C 0:00:30Step 2 65°C 0:00:30

+ Temp Dec. -1.0°C / cycle

Step 3 72°C 0:00:30 x 16


Stage 5 Step 1 72°C 0:05:00 x 1

PROGRAM F:95 RTPCR37

Stage 1Step 1 37°C 1:00:00Step 2 95°C 0:10:00 x 1Hold 4°C


Stage 3 Step 1 72°C 0:05:00 x 1

PROGRAM F:96 RTPCR65

Stage 1Step 1 65°C 1:00:00Step 2 94°C 0:01:00

Stage 2Step 1 94°C 0:00:15Step 2 65°C 0:00:30 x 40

Stage 3 Step 1 65°C 0:07:00 x 1

PROGRAM F:97 RAMPStage 1 Step 1 94°C 0:01:00 x 1

Stage 2

Step 1 94°C 0:00:30 Ramp 1.0°C / Sec

Step 2 55°C 0:00:30 Ramp 1.0°C / Sec

Step 3 72°C 0:00:30 Ramp 1.0°C / Sec

x 30

Stage 3 Step 1 72°C 0:05:00 x 1

PROGRAM F:98 DIGESTStage 1 Step 1 37°C 4:00:00 x 1

PROGRAM F:99 CUT/KIL

Stage 1Step 1 37°C 4:00:00Step 2 95°C 0:15:00 x 1Hold 4°C


11. Appendix 3: Specifications and Ordering Information

Description PxE with 0.5ml Block PxE with 0.2ml BlockCatalogue Number HBPXE05-220/110 HBPXE02-220/110

Block Capacity48 x 0.5ml tubes96 x 0.3ml tubes1 x 96 well plate

96 x 0.2ml tubes1 x 96 well plate

Temperature Control Available

Simulated tube controlSimulated plate controlBlock control

Simulated tube controlSimulated plate controlBlock control

Block Temperature Range 4° C - 99° C 4° C - 99° CBlock Heating Rate Up to 3° C/Sec Up to 3° C/SecBlock Cooling Rate Up to 2° C/Sec Up to 2°C/SecBlock Uniformity ±0.5° C within 30 seconds ±0.5° C within 30 secondsHeated Lid Temperature Range

95° C - 120° C 95° C - 120° C

Display Resolution 0.1° C 0.1°C

PROGRAMMING (all unit variants)Number of programs 99Number of directories 6Maximum number of program stages 10Maximum number of steps per stage 10Maximum programmed dwell time 9 hr 59 minutes 59 secondsTime increment / decrement YesTemp increment / decrement YesTemperature ramping YesPause facility YesAutostart facility YesRun “end time” calculations YesAlphanumeric programming YesPower 550WDimensions (WxDxH) 244 mm (W) x 405 mm (D) x 285 mm (H) Weight 7.2 kg

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User Manual - Hybaid - PXE - HBPXE Thermal Cycler

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