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BXP User Manual - Galileo Equipments

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1. 000000 000 38 6 7 Aborting Programs issi ninna 38 6 8 Program Completion 00 eens eeen 39 CHAPTER 7 TROUBLESHOOTING eecssscsssssssessssssssssssssssssssrsseeeae 41 7 1 Programs Using ACTIVE Tube Control e e00eeee0eee 41 Check the Volume in the Tube Thermistor 00 0 41 Check the Location of Thermistor within the Control Tube 41 Check the Fit of the Tube in the Block 41 Thermo Hybaid Issue 1 0 January 2002 ix Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Check Through POrdduossssuseleisem cecendacatdds nevedeatenaetcancees 41 7 2 Programs Using SIM Temperature Control 42 SIN AD RER EE eae E aai 42 SIA 4 EE 42 ET Se ve 42 Power Katli a 42 7 3 Transfer of Protocols from a Block Control Machine to a Tube Control Machine Using the Thermistor Lead Tate GT EE taco SPI E RO 43 7 4 Optimisation of Protocols seonrnronrronrronnrnnnrnrrrrrerrnrnnnernnnrnrneenne 44 DSH AIAN a Samurai tosentedreeeemanentemucenn tetrade 44 MINE 44 OG ER SEE nr 45 CHAPTER 8 MAINTENANCE hrrvrrvreverrvrnrvrrnrnrrrrrerrrsnressnrserrssernrennrssnrnerresersesnnesenesenn 47 8 1 General Cleaning ccccccsssceessecesssscesssccesseecesseceesneeesseeeees 47 SZ Decorta minty ass A ak a Ala a a a a RNA 47 8 3 Prot
2. Use the RIGHT softkey to move the cursor to NEW and select by pressing ENTER Use the RIGHT softkey to move the cursor to NEW DIR and select by pressing ENTER Use the UP and DOWN cursor to select the directory you wish to rename and press ENTER Use the alphanumeric keypad to enter the new directory name Use the PREVIOUS and NEXT softkeys to move to the next or previous character space Press ENTER to save the new name and return to the main menu Erasing an Existing Directory Programs or complete directories may be erased to vacate program space for future use Select PROG from the Main Menu and then use the RIGHT arrow keys to move the cursor to COPY from the program menu Press ENTER to select Use the LEFT and RIGHT softkeys to move the cursor to ERASE DIR and press ENTER to select Thermo Hybaid Issue 1 0 January 2002 20 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 Select the relevant directory by moving the cursor with the UP and DOWN softkeys and press ENTER 4 A final confirmation screen allows you either KEEP or ERASE the directory 5 3 Programs The programs in the Px2 are divided into stages and steps when displayed on the screen Ina simple PCR program a stage typically includes three steps where each step refers to the temperatures and times within the protocol E g Step 1 95 C for 30 seconds Step 2 55 C for 30 se
3. rtisan Artisan Technology Group is your source for quality TecmologyGrap new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF Instra fg REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED AE FR Contact us 888 88 SOURCE sales artisantg com www artisantg com 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 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888
4. HBPXBG05 HBPX2 O Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Px2 with 0 2ml Gradient Block HBPX2 96 x 0 2ml tubes 1 OmniUltra Plate Tube control Simulated tube control Simulated plate control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 Tube Thermistor 0 2ml HBPXTTM02 HBPXBG02 HBPX2 53 Guaranteed 888 88 SOURCE www artisantg com PROGRAMMING all unit variants Number of programs Number of directories Maximum number of program stages Maximum number of steps per stage Maximum programmed dwell time Time increment decrement Temp increment decrement Temperature ramping Pause facility Autostart facility Run end time calculations File protection Output Alphanumeric programming Gradient software Power Dimensions WxDxH Weight Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation 99 10 10 Ohr 59 mins 59 secs Yes Yes Yes Yes Yes Yes Yes RS232 and RS485 Yes Yes 550W 240mm x 390mm x 280mm 8 6kg 54 Guaranteed 888 88 SOURCE www artisantg com Working Conditions Ambient temperatures of 4 C to 35 C Power requirements 550W at 115 230V a c 10 and 50 60Hz Satisfies the requirements of BS EN 61010 1 1993 NB When the Px2 thermal cycler is removed from a
5. Block Uniformity Heated Lid Temperature Range Display Resolution Ingress Protection Rating Standard Accessories Interchangeable Block Modules Thermo Hybaid Issue 1 0 January 2002 Px2 with In Situ Block HBPX2 4 x microscope slides 76 x 26 x Imm Simulated slide control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 Humidity Chamber HBPXBFB Px2 with 384 Well Block HBPX2 1 x OmniPlate 384 Simulated plate control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 HBPXB384 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 52 Description Catalogue Number Block Capacity Temperature Control Available Performance Block Temperature Range Block Heating Rate Block Cooling Rate Precision Control Block Uniformity Heated Lid Temperature Range Display Resolution Ingress Protection Rating Standard Accessories Interchangeable Block Modules Px2 Chassis Px2 with 0 5ml Gradient Block HBPX2 48 x 0 5ml tubes 96 x 0 3ml tubes 1 x 96 well plate Tube control Simulated tube control Simulated plate control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 Tube Thermistor 0 5ml HBPXTTMO05
6. seconds This is useful when increasing an incubation step to allow for the depletion of an enzyme 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 This type of software control is dependent upon the external thermistor probe located in the microfuge tube This allows very accurate monitoring and control of sample tube temperature This feedback control loop allows the transition to target temperature to be accelerated by creating a temperature gradient between the block temperature and the tube temperature A standard fitting which allows oil free cycling in 0 2 0 3 0 5ml tubes and 96 384 well plates The auto height adjusting heated plate in contact with the top of the reaction vessels maintains the temperature above 100 C preventing evaporation of the sample View The View function allows the contents of programs to be accessed Thermo Hybaid Issue 1 0 January 2002 67 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 68 APPENDIX V Px2 THERMAL CYCLER Pre Set Programs on the Px2 Programs co
7. Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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 QO m 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
8. Issue 1 0 January 2002 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 9 Loading Samples and Heated Lid Operation The Px2 will produce identical cycling profiles whether 1 or 96 samples are to be analysed The following guidelines maximise the uniformity and thermal transfer characteristics of the block thus ensuring optimal sample to sample reproducibility e Load samples uniformly across the block rather than in clusters Spreading the samples spreads the thermal load and helps maintain uniformity e If small numbers of samples are to be run use dummy tubes to ensure that there is at least one tube in each quadrant of the block This facilitates even contact between the heated lid and the tops of the tubes allowing good thermal transfer e Care should be taken when matching block consumable control mode and sample number see Appendix I for a full list of recommended consumables e Ensure tube caps are properly closed before loading the block The heated lid is simple to operate In one action the lid is closed and the heated plate lowered to its correct height and pressure setting 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 4 1 It is defaulted to the tube pressure for the plate pressure turn the knob clockwise until the plate icon is aligned
9. PROGRAM Select PROG from the main menu to access the programming menu Ramp Rate Ramping precisely controls the rate of change of sample Temperature Ramping temperature C sec This is useful for limiting the rate of change of temperature to allow partial extension of short or degenerate primers The RUN Option allows the user to run a stored program on the block When using simulated tube control methods the sample is entered to ensure precise block heating and cooling SET UP This function allows the default settings of the Px2 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 Refer to Section 5 2 for instructions on how to use this control option Simulated Slide Control Simulated Slide Control incorporates an algorithm to reduce the time to target temperature when performing temperature cycling reactions using microscope slides on an Jn Situ block 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 The calculated sample temperature inside sample tubes and block temperature are displayed during the run One stage of a program consists of one or more steps up to a maximum of 10 A stage m
10. with the calculated temperature for each column displayed to the left of the bar e Press BACK to return to the Gradient Calculator screen to enter new parameters or alternatively press MENU to return to the Main Menu screen The Gradient Calculator may be accessed at any time even when the block is active MENU should be pressed to return to the Main Menu and the steps described above should be followed Thermo Hybaid Issue 1 0 January 2002 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 6 Running a Program Design of the Px2 software ensures that running a pre stored protocol is easy At the Main Menu select RUN by depressing the relevant softkey Select the source directory e g F THERMO using the directional UP and DOWN softkeys and press ENTER Select the program number to be run using the number pad or scroll through the programs using the PREVIOUS and NEXT softkeys and press ENTER at the appropriate choice Please note the UP and DOWN keys may be used to advance or return to a parameter if necessary Once the relevant protocol is selected the settings for heated lid operation holdstep loading and end run settings can be defined The PREVIOUS and NEXT keys should be used to scroll between the various possibilities whilst the ENTER key can be used to select the relevant option Lastly the temperature control method is selected by using the PREVIOUS a
11. 20ul 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 Thermo Hybaid Issue 1 0 January 2002 2 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Programming and Operation The combination of new keypad design and large screen display ensure user friendly programming and operation During progr
12. 7 3 Transfer of Protocols from a Block Control Machine to Tube Control Using the Thermistor Lead Extension The different modes of control should be considered before transferring protocols directly E g consider a temperature cycling protocol consisting of 1 minute at 95 C followed by I minute at 65 C repeated 30 times Using block control the actual sample temperature is at 95 C for just 30 seconds at each step a total of 15 minutes overall In contrast tube control will result in precise one minute incubations at each step a total of 30 minutes at the target denaturation temperature Even though tube control gives a more accurate representation of the program transferring such a protocol directly could result in lower yields because the enzyme is exposed to the high temperature for significantly longer thus reducing its activity in later cycles The most accurate way to transfer protocols is to use the control tube of the Px2 as a temperature probe in the block control machine as follows 1 Set the Px2 to run a single program at a set temperature for an extended time e g 37 C for 4 hrs ensuring that the heated lid is switched off and the instrument is set to block control This enables the tube thermistor to be used remotely 2 Connect the thermistor lead extension to the thermistor control tube and plug it into the Px2 3 Place the thermistor tube probe into the corresponding thermistor probe socket of the Px2 and loca
13. 88 SOURCE www artisantg com Px2 THERMAL CYCLER PCR License Registration Authorised Thermal Cycler This instrument Serial NO oe eeeeeseeeeseeeeeeeeeeeeeneees 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
14. C sec to 9 99 C sec with the maximum practical setting being 3 00 C sec with current technologies The data entered refers to the ramp rate to the CURRENT step from the PREVIOUS temperature set point For example cycle sequencing of some targets requires the use of degenerate primers and a ramp rate set at 1 C second between the annealing temperature and extension temperature 5 5 Gradient Feature This feature is only available to those who have purchased a 0 2ml or 0 5ml gradient block for temperature gradient cycling The gradient feature is designed to allow the rapid optimisation of annealing parameter between 30 70 C and can be accessed through the Advanced Edit function see section 5 4 The minimum temperature to be assessed is entered along with the gradient spread maximum of 15 C After a gradient spread value has been entered in the GRAD section the number will be displayed on the main programming screen E g lt G 15 gt Thermo Hybaid Issue 1 0 January 2002 28 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Gradient Programming Worked Example 1 In the EDIT mode NEW or EXISTING press ENTER to move to the relevant annealing temperature step The annealing temperature entered in this step should be the lowest temperature within the gradient You must enter a time value as well in order to access the gradient function in the Advanced Edit scree
15. It is possible to edit programs whilst the Px2 is running a different protocol 1 To edit and overwrite existing programs select PROG from the Main Menu using the appropriate softkey 2 Select EDIT from the Program Menu by pressing ENTER 3 Select EXISTING from the Program Editing Menu by pressing ENTER 4 Use the UP and DOWN keys to move the cursor to the appropriate source directory and press ENTER to select The BACK button may be used to return to the previous screen if necessary 5 The keypad numbers or PREVIOUS and NEXT softkeys can be used to select the number of the program to be edited Press ENTER to select the program 6 Use the alphanumeric keypad in combination with the PREVIOUS and NEXT softkeys to edit the program name Press ENTER to proceed or BACK to return to the previous screen 7 Use ENTER to advance through the fields and the LEFT softkey to move across the menu Time and temperature parameters can be altered using the numerical keys as required The UP softkey can be used to move the cursor back to previous screens Note To increase the number of steps in a stage simply input values into the zero time temperature fields at the end of a stage To decrease the number of steps in a stage just input the new data over existing time temperature values 8 To complete editing stage 1 press ENTER with each parameter set at zero Thermo Hybaid Issue 1 0 January 2002 23 Artisan Technology Group Quality Inst
16. after 90 minutes the CONTINUE key has not been pressed to start a program a manual start time out screen is displayed and the run is cleared Thermo Hybaid Issue 1 0 January 2002 38 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com No Heated Lid If the heated lid 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 NB The Lid Heating indicator light will remain off n this mode and the Ready Working light will come on immediately 6 6 Hold amp Pause Function When entering a program the Px2 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 The screen will display HOLD during a run If a Hold is inserted between stages the program can be advanced to the next stage by pressing CONTINUE If a Hold is inserted at the last stage pressing CONTINUE will switch to the run summary screen Common uses of the Hold step include the following e Inserting an initial 95 C incubation at the start of the protocol to perform the Hot Start procedure After the enzyme has been added pressing CONTINUE will advance the program into the cycling part of the protocol e A final low temperature 4 10 C hold for the end of overnight runs
17. 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 Thermo Hybaid Issue 1 0 January 2002 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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
18. in this case the control algorithm has been adjusted so that the programmed temperature is achieved on the top surface of a standard microscope slide The performance of the Jn Situ module has been measured using miniature thermistor probes attached to the surface of slides Optimum results in terms of overshoot and uniformity across four standard glass microscope slides 0 8mm 1 0mm thickness with the humidity chamber in place have been obtained using a calibration factor of 25 the default value Using a higher calibration accelerates the approach to target temperature but results in some degree of overshoot Conversely a lower calibration factor slows the approach to target temperature Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Block Control Controls block temperature as on a conventional dry block machine We do not recommend this means of control for thermal cycling due to the variability in thermal profile obtained with different sample volumes and consumable types 6 5 Heated Lid Preheat Once a program and block have been selected and calibration factors entered where appropriate the program will proceed according to the setup conditions as outlined below The heated lid typically takes around 2 minutes to reach operating temperature During this time the block is controlled at 4 C WARNING Both the tops of r
19. least 5 times Repeated washing and autoclaving of the Mats causes them to crack around the edges When this starts to happen they should be discarded NB The number of times an OmniSeal TD Mat can be reused is reduced considerably if it is cleaned with alcohols or detergents or if exposed to UV irradiation Limitation of Microtitre Plate Operations Some users have reported loss of volume when using microtitre plates for thermal cycling Recent experiments performed at Thermo Hybaid have demonstrated that volume loss is independent of sample volume and occurs through the sides of the polycarbonate microtitre plate all manufacturer plates tested and is not due to poor sealing Typically volume loss of 3ul per well in a 30 cycle reaction can be expected The Thermo Hybaid OmniUltra helps to address this problem to some extent In our tests reaction volumes of 101 gave strong results Sul reaction volumes gave visible but faint results thus 10ul is the minimum recommended sample volume for this plate This is because this plate is injection moulded which results in absolute well to well and plate to plate consistency The well wall thickness of conventional vacuum moulded plates is inconsistent resulting in highly porous patches which allow large amounts of sample to pass through If you are working with sample volumes of less than 10ul and are using the OmniUltra it may be advisable to switch to polypropylene consumables instead For
20. 4 Move the cursor using the UP and DOWN softkeys to select the target directory and press ENTER Thermo Hybaid Issue 1 0 January 2002 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 Scroll through the possible program locations using the PREVIOUS and NEXT softkeys and press COPY All empty file locations are marked as empty It is possible to overwrite full program locations A confirmation screen checks whether you wish to overwrite the data press COPY or ABORT as desired Erasing an Existing Program Programs or complete directories may be erased to vacate program space for future use 1 Select PROG from the Main Menu and then use the RIGHT arrow key to move the cursor to ERASE from the program menu Press ENTER to select 2 Again use the RIGHT arrow key to move the cursor to ERASE PROG and press ENTER to select 3 Select the directory containing the relevant program by moving the cursor with the UP and DOWN softkeys and press ENTER 4 Use the PREVIOUS and NEXT keys to choose the relevant program and press ERASE 5 A final confirmation screen allows you either KEEP or ERASE the program 5 4 Advanced Edit Features The Advanced Edit features allow the creation of more complex cycling protocols in order to enhance experimental data It is possible to program e An incrementation or decrementation of both time and temperature on a cycle by cycle basis e
21. 6 0 00 30 0 00 30 0 00 30 x 5 0 05 00 x 1 70 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com PROGRAM F 95 Stage I Stage 2 Stage 3 PROGRAM F 96 Stage I Stage 2 Stage 3 PROGRAM F 97 Stage I Stage 2 Stage 3 RTPCR37 Step I Step 2 Hold Step I Step 2 Step 3 Step I RTPCR65 Step I Step 2 Step I Step 2 Step I RAMP Step I Step I Step2 Step 3 Step I O Thermo Hybaid Issue 1 0 January 2002 37 C OSE 4 C 95 C SC TY 72 C 65 C 94 C 94 C 65 C 65 C 94 C 94 C 55 C 12 C 12 C 1 00 00 0 10 00 0 00 30 0 00 30 0 00 30 0 05 00 1 00 00 0 01 00 0 00 15 0 00 30 0 07 00 0 01 00 0 00 30 0 00 30 0 00 30 0 05 00 x I x 40 x I x 40 x I x 1 Ramp 1 0 C Sec Ramp 1 0 C Sec Ramp 1 0 C Sec x I 71 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com PROGRAM F 98 DIGEST Stage I Step I 37 C 4 00 00 x 1 PROGRAM F 99 CUT KIL Stage I Step I 37 C 4 00 00 Step 2 95 C 0 15 00 x 1 Hold 4 C Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 72 rtisan Artisan Technology Group is your source for quality TecmologyGrap new and certified used pre owned equipment FA
22. Although unnecessary for the vast majority of protocols some scientists prefer to have this step included e A final 72 C incubation to ensure completion of the final extension step of a reaction Pressing PAUSE during a cycling program will pause the program at the current or next target temperature within the step Pressing CONTINUE will continue the countdown for the step 6 7 Aborting Programs The program will run to completion unless interrupted by the pressing of either the PAUSE or STOP keys whilst the appropriate run screen is displayed Pressing the STOP key once will display a verification screen the program continues while this is displayed Pressing ABORT aborts the program and returns you to the Main Menu screen Pressing CONTINUE returns you to the Run screen and continues the run Thermo Hybaid Issue 1 0 January 2002 39 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 6 8 Program Completion At the end of the program the END OF RUN screen will be displayed In this case the heated lid will be switched off automatically The alarm if selected will sound and the block will remain at 20 C for 30 minutes to drive the heated lid plate temperature to ambient If a final HOLD temperature has been specified the heated lid if selected will remain on For most thermal cycling applications a final HOLD at elevated temperature is not necessary Extended hig
23. SOURCE www artisantg com 50 Description Catalogue Number Block Capacity Temperature Control Available Performance Block Temperature Range Block Heating Rate Block Cooling Rate Precision Control Block Uniformity Heated Lid Temperature Range Display Resolution Ingress Protection Rating Standard Accessories Interchangeable Block Modules Px2 Chassis CHAPTER 9 Px2 THERMAL CYCLER Technical Specifications amp Ordering Information Px2 with 0 5ml Block HBPX2 48 x 0 5ml tubes 96 x 0 3ml tubes 1 x 96 well plate Tube control Simulated tube control Simulated plate control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 Tube Thermistor 0 5ml HBPXTTMO05 HBPXBO5 HBPX2 Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Px2 with 0 2ml Block HBPX2 96 x 0 2ml tubes 1 OmniUltra Plate Tube control Simulated tube control Simulated plate control Block control 4 C 99 C Up to 3 C Sec Up to 2 C Sec 0 1 C 0 4 C within 15 secs 95 C 120 C 0 1 C 20 Tube Thermistor 0 2ml HBPXTTM02 HBPXB02 HBPX2 51 Guaranteed 888 88 SOURCE www artisantg com Description Catalogue Number Block Capacity Temperature Control Available Performance Block Temperature Range Block Heating Rate Block Cooling Rate Precision Control
24. ST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF Instra fg REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED AE FR Contact us 888 88 SOURCE sales artisantg com www artisantg com
25. The rate at which the temperature changes between two given temperatures e Gradient steps for gradient compatible blocks Advanced Edit features are accessed by pressing the ADVANCED softkey at the temperature step at which the function is to take effect If Advanced Edit data already exists for any step lt ADV gt is indicated on the screen Thermo Hybaid Issue 1 0 January 2002 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Enter and change Advanced Edit parameters using the number arrow and ENTER keys The Advanced Edit feature can also be accessed from the normal EDIT program menu options if Advanced Edit parameters are to be added to an existing program If desired the Advanced Edit feature can be deleted for the current step To do this set all the parameters back to zero The lt ADV gt message will then not be shown in the temperature time set up screen denoting that no Advanced Edit functions are operating Time Advanced Edit Worked Example The user can specify the increment decrement per cycle for each program stage Time increments can be used for example in high cycle number reactions to allow longer for enzyme action with successive cycles Below 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 act
26. This symbol indicates high voltage Risk of electric shock gt gt Be AVERTISSEMENT Risqu de choc l ctrique In addition the following conventions are adopted in the manual in respect of indicating safety hazard A 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 Thermo Hybaid Issue 1 0 January 2002 8 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com CHAPTER 3 Px2 THERMAL CYCLER Unpacking amp 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 Px2 Control Chassis Px2 Block Module optional User Instruction Manual Mains Lead Control Thermistor Probe 0 2ml and 0 5ml gradient and standard block modules Control Th
27. ammed 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
28. 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 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 When installing removing the interchangeable block e Ensure that the power to the instrument is turned off e The Block Heat Sink may be hot after use Use gloves when changing blocks if the unit has been used recently 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 Thermo Hybaid Issue 1 0 January 2002 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 2 2 Symbols amp Conventions The Px2 has been designed for safe operation The following symbols appear on the unit and their meanings should be noted Indicates the ON position of the main power switch 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
29. ape are unique single use sealing systems designed for oil free thermal cycling in 96 well plates and to prevent cross contamination between wells After a thermal cycling reaction OptiTape or TDX Tape can be removed from the plate with ease OptiTape Supplied on a roll with optional dispenser and designed for use with Thermo Hybaid s OmniUltra Plate although will give an exceptional seal with other brands of polycarbonate plate TDX Tape Clear in colour and designed for use with polypropylene plates such as OmniTube 96 OmniFast 96 or OmniFast 384 OptiTape and TDX Tape cannot be interchanged as the TDX Tape will bind irreversibly to polycarbonate To secure contact with the heated lid of the thermal cycler the OptiTape and TDX Tapes must be used in combination with the reusable TD Compression Pad Sealing a 96 Well Plate 1 The tape can be applied to 96 well plates by positioning over one of the short edges and peeling back the lining material allowing the adhesive layer to fall on to the plate This should be possible without introducing creases However before heating the adhesive is soft enough to be repositioned without adverse effects Thermo Hybaid Issue 1 0 January 2002 59 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 2 The tape should be firmly anchored by pressing over each well This can be done using a soft roller or by finger pressure Espe
30. ate plate e 1x OmniFast 96 well skirted polypropylene plate e 1x OmniTube 96 single piece polypropylene plate e 96 x 0 2ml OmniStrip tubes e 96 x 0 2ml individual OmniTubes The 0 5ml Standard and Gradient Block Module This block can hold the following e 48 standard 0 5ml individual OmniTubes The Flat Block Module This block accommodates up to four standard microscope slides Thermo Hybaid Issue 1 0 January 2002 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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 lt
31. ay be repeated for temperature cycling Up to ten stages may be used in one program and stage to stage linking is automatic Thermo Hybaid Issue 1 0 January 2002 66 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sub ambient Block TEMP INC DEC Temperature Increment Decrement TIME INC DEC Time Increment Decrement Touchdown PCR TUBE Active Tube Control HEATED LID 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 The aluminium block used on sub ambient Px2 systems This uses Peltier devices for heating and cooling Operating range 4 C to 99 C 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 A thermistor is a resistor whose resistance changes with temperature It can therefore be used as a very accurate temperature probe with small liquid volumes 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
32. be for 0 5ml blocks NB Do not disconnect a tube thermistor when a program utilising tube control is in progress If this does occur the program will be abandoned and an error message will be displayed NB Tube thermistors from TouchDown OmniGene and Omn E instruments are not compatible and CANNOT be used with Px2 thermal cyclers Extension Lead for the Tube Thermistor The Px2 is provided with an extension lead for the thermistor This should only be used for transferring protocols from instruments that cannot use Active Tube Control See Section 7 3 for further details Thermo Hybaid Issue 1 0 January 2002 35 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Simulated Tube Control SIM TUBE This temperature control method uses an algorithm similar to tube control However with simulated tube control the block temperature overheat characteristic which is used to eliminate the sample temperature lag is based on calculated values rather than the temperature monitored by the tube thermistor Similar considerations apply when transferring protocols from a block control machine as discussed above and the extension lead can again be used Reactions using Simulated Tube Control do not require the tube thermistor to be connected but sample loading and volume details must be entered when prompted by the run screen For all tube reactions in a 0 2ml or a 0 5ml thin wall
33. cially with the OptiTape ensure that no air pockets can be seen between the tape and the plate 3 Once in the machine the Compression Pad HB TD SFOAM must be placed 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 Place the sealed 96 well plate on the machine Close the lid and select the Plates icon Note if an OmniTube 96 Plate is being used the tubes icon should be selected as the heated lid setting Removing the Tape Prior to removing the Tape or OmniSeal Mat from a plate ensure there is no condensation of the solution inside the wells or on the plugs of the tape by either 1 Removing the 96 well plate from the block and cooling to 4 C 2 Cooling down the PCR block to 4 C if possible 3 Heating the sample to 35 C for 30 minutes This will minimise the risk of well to well contamination when removing the Tape from the 96 well plate Tape is most easily removed while the 96 well plate is in a rack or still on the machine The sheet should be peeled back from one corner diagonally across the plate using a single smooth action Resealing Using OptiTape OptiTape is designed for single use Once heated and compressed the backing conforms to the plate and cannot be easily repositioned Reuse is therefore not advised Thermo Hybai
34. cold room it should be left to equilibrate for 2 3 hours to avoid condensation Thermo Hybaid Issue 1 0 January 2002 55 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 56 APPENDIX I Px2 THERMAL CYCLER Recommended Consumables Size i Systems Opti Tape with 100 Sealing tape for OmniUltra Plate or other mer 100 HB TCT 100 polycarbonate plates Includes universal dispenser HB TCT 250 OptiTape Refill 100 Refill roll of OptiTape sealing sheets for HB TCT 100R Dispenser OmniUltra or other polycarbonate plates HB TCT 250R TDX Tape Sealing tape for polypropylene plates HB TD TAPE100 1 Compression Foam pressure pad for use with tape sealing HB TD SFOAM Pad system Reusable 96 well sealing mat HB TD MTSRS5 Mat Plasticware Size ms OmniUltra 25 Polycarbonate 96 well plate robot compatible HB TC 25D Non Irradiated OmniUltra Gamma 25 Polycarbonate 96 well plate robot compatible HB TC 25N Irradiated 5 Thermo Hybaid Issue 1 0 January 2002 57 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com RN Se OmniTube 0 2ml 1000 0 2ml individual tubes with integral domed caps HB TC 3372N OmniTube 0 2ml 1000 0 2ml individual
35. conds Step 3 72 C for 30 seconds In addition each stage can be repeated up to 99 cycles and a hold added to the end of the stage The Px2 allows you to program up to 10 separate stages each with up to 10 separate steps This means that even the most complex thermal cycling protocol may be saved in a single program space Advanced editing features adjusting ramp speeds setting a gradient of annealing temperatures changing time temperature with successive cycles can be accessed by pressing the ADVANCED softkey at the relevant step see section 5 4 for further details Creating a New Program 1 From the Main Menu select PROG using the appropriate softkey 2 Move cursor to EDIT then press ENTER to select 3 Move the cursor to NEW then press ENTER to select 4 Move the cursor to NEW PROG then press ENTER to select 5 Move the cursor to desired target directory and press ENTER to select 6 The instrument allocates a program number of the next available program space You are now able to enter the user or protocol name using the alphanumeric keypad Use the PREVIOUS and NEXT softkeys to move to the position of the cursor where the character is to be added Press ENTER only when the name is complete If no characters are entered the default NO NAME will be entered The BACK button may be used to return to the previous screen if necessary Thermo Hybaid Issue 1 0 January 2002 21 Artisan Technology Group Qual
36. d Issue 1 0 January 2002 60 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com OmniSeal TD Mat Cleaning amp Sterilisation Procedures The OmniSeal TD Mat Catalogue No HB TD MT SRS 5 is used to seal 96 well plates when using the Px2 heated lid Mats can be cleaned and sterilised using the procedures described below Soak for I minute in either 10 hypochlorite solution or 0 1M hydrochloric acid solution and then rinse with distilled water Dry inside an oven at up to 80 C if required Autoclave at 121 C for 15 minutes Sealing a 96 Well Plate OmniSeal TD Mats have 96 plugs in the underside They are spaced so that they fit into the wells of a 96 well plate The plugs are shaped to expand sideways in order to seal all 96 wells of a 96 well plate when sufficient pressure is exerted to the upper surface of the mat by the Px2 heated lid Figure 10 1 Showing how the OmniSeal TD Mat seals the wells of a 96 well plate OMNISEAL TD PRESSURE 96 WELL PLATE WELL Removing the OmniSeal TD Mat To minimise the risk of well to well contamination remove the OmniSeal TD Mat from the 96 well plate whilst the plate is in a rack or on the machine Thermo Hybaid Issue 1 0 January 2002 61 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Reusability of the OmniSeal TD Mats The OmniSeal TD Mat can be reused at
37. ded into the F HYBAID Directory of the Px2 memory can be used directly or as building blocks for other protocols using the COPY function PROGRAM F 90 3T_PCR Stage I Step I 94 C 0 01 00 x 1 Stage 2 Step I 94 C 0 00 30 Step 2 55 C 0 00 30 Step 3 72 C 0 00 30 x 30 Stage 3 Step I 72 C 0 05 00 x 1 Hold 4 C PROGRAM F 91 2T_PCR Stage 1 Step I 94 C 0 01 00 x 1 Stage 2 Step I 94 C 0 00 10 Step 2 68 C 0 00 10 x 30 Stage 3 Step I 72 C 0 05 00 x I Hold 4 C PROGRAM F 92 LONGPCR Stage 1 Step 1 94 C 0 02 00 x I Stage 2 Step I 94 C 0 00 10 Step 2 65 C 0 00 30 Step 3 68 C 0 10 00 x 10 Stage 3 Step I 94 C 0 00 10 Step 2 65 C 0 00 30 Step 3 68 C 0 10 00 Time Inc 0 20 cycle x 20 Stage 4 Step I 68 C 0 07 00 x I Thermo Hybaid Issue 1 0 January 2002 69 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com PROGRAM F 93 Stage I Stage 2 PROGRAM F 94 Stage I Stage 2 Stage 3 Stage 4 Stage 5 CYCSEQ Step I Step I Step 2 Step 3 Hold TD PCR Step I Step I Step 2 Step 3 Step I Step 2 Step 3 Step I Step 2 Step 3 Step I O Thermo Hybaid Issue 1 0 January 2002 94 C 94 C 50 C 60 C 10 C 94 C 94 C 65 C 12 C 94 C 65 C 72 C 94 C 50 C 12 C TE 0 01 00 x I 0 00 10 0 00 10 0 04 00 x 25 0 01 00 x 1 0 00 30 0 00 30 0 00 30 x4 0 00 30 0 00 30 Temp DEC 1 0 C cyck 0 00 30 x 1
38. e Thermistor Positioning The control thermistor probe when required should be inserted into socket A2 to the left of the block towards the rear of the instrument see fig 3 2 Ensure that the tube contains the same volume of mineral oil as the sample reaction volume and that the probe is immersed in the oil and not touching the wall of the reaction vessel Figure 3 2 Well Numbering Showing Tube Thermistor Positioning in A2 Use in Cold Rooms The Px2 may be used in a cold room down to 4 C However when removed to room temperature the instrument should be allowed to equilibrate for at least two hours before being switched on If equilibration is not done condensation may form within the unit and cause a short circuit Thermo Hybaid Issue 1 0 January 2002 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 12 CHAPTER 4 Px2 THERMAL CYCLER Operating Instructions 4 1 Initial Start Up a Switch on the Px2 using the power switch at the rear of the unit When first switched on the unit will complete a start up and self test routine b After 5 seconds a screen will display Self check procedure in progress The self test routine checks the block thermistor heating and cooling circuits and the heated l
39. e Thermo Hybaid 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 The use of 35S labelled nucleotides is not covered under our warranty agreement and requires special service arrangements 8 3 Protection to the User The Px2 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 8 4 Protection of the Instrument Fuses The Px2 mains power inlet is fitted with two T6 3A fuses 20mm x 5mm If necessary these may be replaced by a qualified person 8 5 Tube Thermistor Care The tube thermistor assembly ensures that the programmed temperature profile is accurately and reproducibly achieved within all sample tubes As a sample mimic it is important that the thermistor Thermo Hybaid Issue 1 0 January 2002 48 Artisan Technology Group Quality Inst
40. e 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 Thermo Hybaid Issue 1 0 January 2002 y Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com vi Px2 THERMAL CYCLER Contents PCR LICENSE REGISTRATION 2 si una s ln sua a ii P DIAGRAM oid Sate eee ee es ee eee iv WARRANTY EE a kirna ninna v CHAPTER 1 INTRODUCTION negar 1 R R 0300 0 SS RN OR 1 2 P32 SA 1 The 0 2ml Standard and Gradient Block Module 00 0e 00000000 1 The 0 5ml Standard and Gradient Block Module 00 0e 0000 00 1 The Flat Block WMO iss in EN 1 The 384 Well Block Module ronnrnrrnnoonnvnrnnrvnerrrneenrnvnnnrrnsnnrrvsnnrnvennnne 2 Temperature Control Methods is cccc seceedecestccdsncsasteedsseeacceseswsdedeenedeetans 2 rd vvs 2 Programming and Opera aaa ia a al aaa 3 Pre set Prostam ve 3 Advanced Edit Temperature Ramping eseeeeeeeseeeseeeeeeeeeeeeneees 3 Advanced Edit Time Temperature Increment ronorrrnvnnnrrrvvrvrnvnnvnrnenn 3 Ad
41. e heated lid should always be closed during temperature cycling even if the lid is not switched on The Px2 is intended for indoor use at an ambient temperature of 4 34 C in conditions of up to 80 humidity These specifications have been calculated for operations at between 0 and 2000m altitudes Block Installation and Removal 1 Remove the Px2 Control Chassis and Block Module from the packaging and place on the bench do not connect to the mains supply immediately Open the lid by turning the knob anticlockwise to the open lid icon to expose the block recess Supporting the block from the blue plastic moulding and the ring pull position the back of the block on the two lugs on the chassis Lower the block via the pull ring into the recess see fig 3 1 Placing your thumb on the ring pull just beneath the hole firmly push downwards until you hear the block click into place Do not force the block if this does not happen remove the block and check for obstructions in the block recess The following error screens will appear if the block has not been installed properly or you attempt to run a program without a block in place SELF CHECK FAILED or SELF CHECK FAILED BLOCK NOT PRESENT BLOCK ID ERROR Removal is the reverse procedure Thermo Hybaid Issue 1 0 January 2002 10 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Figure 3 1 Installing the Block Modul
42. eaction 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 Heated Lid Automatic Start This should be selected for robust cycling reactions where the reactants can withstand being incubated for the duration of the preheat about 2 minutes without the generation of spurious products After the program is selected the Lid Heating indicator light will switch on denoting preheating of the lid When the lid reaches operating temperature the Ready Working indicator light will change to green and the temperature cycling will start automatically In this mode samples should be loaded and the lid closed before starting the preheat Heated Lid Manual Start This should be used for sensitive cycling reactions 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 After the program is selected the Lid Heating indicator light will switch on denoting preheating of the lid When the lid reaches operating temperature the Ready Working indicator light will change to green An alarm will sound if activated and the START key will need to be pressed to start temperature cycling In this mode samples should be mixed loaded and the lid closed after the preheat is completed If
43. ection to the User ij ccsisscccscasccsccvascasesecacdceascaseedceassvecetasseetacaeive 48 8 4 Protection of the Instrument soooonononoronnrnonrrrrrrrerrrrnnrnrnrnrrnneenne 48 gl EE EEE canoes 48 8 5 Tube Thermistor Care 48 CHAPTER 9 TECHNICAL SPECIFICATIONS amp ORDERING INFORMATION 0000 51 APPENDIX I RECOMMENDED CONSUMABLES eee 57 APPENDIX II SEALING SYSTEMS THE OMNISEAL TD MAT amp TD TAPE 59 APPENDIX III IN SITU BLOCK APPLICATION NOTES r 63 APPENDIX IV GLOSSARY OF TERMS 0 0 00 65 APPENDIX V PRE SET PROGRAMS ON THE Px2 0000 eee 69 Thermo Hybaid Issue 1 0 January 2002 x Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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 e 1x OmniUtra 96 well skirted polycarbon
44. ed tube the volume factor is the total reaction volume in ul in one well including any oil overlay Simulated Plate Control SIM PLATE This mode of control operates on a similar principle to simulated tube control Gee Figure 6 1 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 system is optimised for the 0 2ml block with thin walled polypropylene plate Figure 6 1 Modes of Control used with the Px2 a Active Tube Control 8 a GO FASET N s an SENN PTT TTT HH PTT TTT TTT TET TT Temperature C a Seeeers 3 TRETT TTTTELITTETTTTTE Time secs Block mmm Sample Program 94 C 30 secs Thermo Hybaid Issue 1 0 January 2002 36 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com b Simulated Control Tube Plate Slide Temperature C vi TJ L me ttt ttt SSS 05000 H SaR ARRABSAABSSRLBBRRES Seon Time secs Block mmm Sample Program 94 C 30 secs c Block Control Temperature oC NAT JET ER A LANDE SED SER an w wen DOD De e N w wn vo oneness 22322 228888 aa Time Secs Block aman Sample Program 94 C 30 secs Simulated Slide Control SIM SLIDE This mode of control operates on a similar principle to simulated tube control However
45. ermistor Probe Extension Lead 0 2ml and 0 5ml gradient and standard block modules Specific Consumable Pack RS232 Lead OS ae Wee oo x NB The Jn 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 Thermo Hybaid Issue 1 0 January 2002 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 3 Installation Choosing a Location 1 Where possible avoid connecting the Control Chassis to a mains supply subject to mains voltage fluctuations e g a socket shared by an ultracentrifuge or refrigerator In Px2 systems excess heat is removed from the system by a fan The air intake for the fan is located underneath the unit and air is expelled out of the back of the unit To ensure correct airflow around the unit allow at least 8cm clearance between the sides of each module and 15cm of clearance at the back of the unit Special care should be taken not to obstruct the vent underneath the unit with for example loose Benchcote or sheets of paper The unit must not be covered during operation All Px2 instruments should be placed on a stable and level surface out of direct sunlight and away from strong currents of hot or cold air Th
46. g and operation of the In Situ module are identical to the standard Px2 tube modules see Chapter 5 with the exceptions previously noted and also no heated lid operation is supported For simple fixed temperature incubations of the slides manual control can be used see section 4 7 For more complex multistep incubations or for temperature cycling reactions a protocol can be designed and run using Simulated Slide Control see Chapter 6 Humidity Chamber The polycarbonate Humidity Chamber has been designed to reduce drying out of the sample on the slides For accurate temperature performance the In Situ module should be used with the Humidity Chamber in place and the lid closed e Fill the reservoirs with water e Pre heat the block with the Humidity Chamber in place This allows the reservoir to warm up so that evaporation of the solution can begin Alternatively preheat the reservoir solution to the incubation temperature required e For thermal cycling with extended high temperature incubations it is important to seal the reagents under the cover slip Thermo Hybaid recommends the use of EasiSeal sealing frames These are available in three sizes 25ul HB OS SSEZI1E 65ul HB OS SSEZ2E and 125ul HB OS SSEZ3E They will completely seal the specimen area to prevent drying NB If a petroleum based rubber compound is used for sealing cover slips in place the compound should be allowed to dry out completely before use with this syste
47. h temperature hold steps can lead to evaporation and condensation problems particularly with polycarbonate plates Thermo Hybaid Issue 1 0 January 2002 40 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 41 CHAPTER 7 Px2 THERMAL CYCLER Troubleshooting 7 1 Programs Using ACTIVE Tube Control Check the Volume in the Tube Thermistor Check the volume of mineral oil in the control tube a vial of suitable mineral oil is provided alternatively Sigma molecular biology grade mineral oil M5904 or equivalent can be used Do not use aqueous solutions in the control tube as this can reduce the life expectancy of the thermistor Too much oil in the control tube will result in overshoots in the sample tube temperatures whilst too little oil there will result in a time lag in the samples achieving temperature The volume of oil in the control tube should match that in the reaction tubes including any oil overlay Repeated overshoots at the denaturation temperature during temperature cycling will reduce the activity of the thermostable enzyme resulting in poor yields of product The correct position for the control tube is position A2 Check Location of Thermistor within the Control Tube Always ensure the thermistor probe is located cent
48. he last stage entering values of 0 0 will again bring access to the number of cycles required screen Enter the number of cycles Enter a hold temperature if required If a hold temperature is not entered the instrument will cool to room temperature once the protocol is finished When ENTER is pressed a new STAGE and STEP is displayed By ENTERING values of 0 0 in the first step of a new stage the SAVE screen is accessed 12 Press SAVE to save the program or DISCARD to abort the program Pressing MENU at any time aborts the programming Editing an Existing Program Thermo Hybaid Issue 1 0 January 2002 22 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Existing programs can be altered or overwritten by using the EDIT function Please note that changes made are permanent and the original program is not retained If the original program is still required the COPY function should first be used to save the program to a different program space see Section 5 3 4 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 amending the current entry Advanced editing features adjusting ramp speeds setting a gradient of annealing temperatures changing time temperature with successive cycles can be accessed by pressing the ADVANCED softkey at the relevant step see section 5 4
49. 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 e The Px2 has a power input socket located on the back of the unit Thermo Hybaid Issue 1 0 January 2002 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com e 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 Ema
50. id This takes approximately 30 seconds As the block will become hot during the self test procedure please keep the lid closed 4 2 Main Menu After successful start up the first menu to be displayed is the Main Menu 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 4 1 Summary of Main Menu Functions Select RUN to run an existing program Select MAN to run a manual static incubation Select PROG to edit copy view or erase a program Select SET UP to alter the time date and power resume function and display contrast settings and to identify software version fitted Select CALC to access the gradient calculator software only relevant to gradient protocols Selection of the alternative Main Menu functions is achieved by depressing the soft key directly beneath the relevant display These functions are discussed fully in the following chapters Thermo Hybaid Issue 1 0 January 2002 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 3 Operating Keys The Px2 software is designed to be intuitive the following table gives a summary of the primary functions of each of the operating keys Table 4 2 Function Keys ENTER KEY Selecting Menu and
51. il globalsupport thermohybaid com Alternatively appropriate contact details for your local Thermo Hybaid subsidiary or authorised distributor are provided on our website www thermohybaid com Thermo Hybaid Issue 1 0 January 2002 6 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com CHAPTER 2 Px2 THERMAL CYCLER Safety Precautions 2 1 General Safety Precautions 1 10 11 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 Before use ensure that the unit has been set to the appropriate mains voltage 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 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 mains supply and contact an authorised service centre 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 The tops of the reaction vessels
52. in the range 70 75 C see data sheet from manufacturer The time required depends on the length of product being synthesised Thermo Hybaid Issue 1 0 January 2002 45 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 46 CHAPTER 8 Px2 THERMAL CYCLER Maintenance 8 1 General Cleaning Before using any cleaning or decontamination method except those recommended below please contact your local Thermo Hybaid office or authorised dealer to check that the proposed method will not damage the equipment 1 All surfaces of the Px2 system and heated lid should be cleaned regularly with a soft cloth hot water and a mild detergent 2 Itis important to thoroughly dry all surfaces after cleaning 3 The Px2 is not intended for use with aggressive chemicals and on no account should organic solvents be used to clean this equipment 4 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 performance 8 2 Decontamination When 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 microtitre plates thus contaminating the block and
53. ith successive cycles This example shows 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 Stage 1 Enter parameters to create 95 C 30s 65 C 30s x 4 Cycles 72 C 30s Stage 2 Enter parameters to create 95 C 30s 65 C 30s 1 0 C cycle x 16 Cycles 72 C 30s Stage 3 Enter parameters to create 95 C 30s Thermo Hybaid Issue 1 0 January 2002 27 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 50 C 30s x 5 Cycles 72 C 30s 1 In the EDIT mode NEW or EXISTING move to the temperature step of interest Stage 2 Step 2 2 Press the ADVANCED softkey to access the Advanced Edit screen Navigate through the screen until you reach the TEMP field using the softkeys Use TOGGLE to alternate between Increase INC and Decrease DEC in set point temp cycle Enter the desired value by which the set point temp per cycle should change C using the number keys minimum change is 0 1 C per cycle 3 Move back to the step screen using the ENTER or the BACK lt ADV gt will now appear on screen Changing Ramp Rates Ramp rates can be altered in the Advanced Edit screen The default ramp rate 0 00 C sec is as fast as possible The settable range is 0 01
54. ity Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note If there are no free program spaces the Px2 will prompt you to overwrite an existing program 7 Enter temperature e g 95 C and time hr min sec as required Use the LEFT softkey to move the cursor as desired or ENTER to advance to the next step The UP softkey can be used to scroll backward to correct a mistake The program will not allow you to advance to step 2 until a valid temperature and time have been entered Note Pressing the ADVANCED softkey at any step throughout the programming provides access to the advanced edit features These facilitate the creation of more complex protocols including touchdown or gradient methods see section 5 4 for more details 8 Continue to enter the desired time and temperature STEPS of this STAGE If no more STEPS are required in the STAGE press ENTER leaving each parameter at 0 0 until you access the number of cycles screen 9 Enter the number of times you would like to cycle through the STAGE using the alphanumeric keys A hold step can also be programmed after each stage so once the cycles have been completed the Px2 will wait at the specified temperature until manually advanced This is particularly useful for hot start protocols If a hold step is not required press ENTER to reach the next stage 10 Continue adding steps to the new stage as per stage 1 11 When all the steps have been entered in t
55. ity of the mineral oil In addition PCR buffer can often cause corrosion of the thermistor thus reducing the life expectancy of the probe The thermistor acts as a sample mimic monitoring the sample temperatures as it changes during cycling feeding back this information to the Px2 processor This feedback allows the unit to respond to the sample temperature ensuring that the samples achieve the exact temperatures and times programmed To bring the sample to temperature rapidly the block is heated cooled beyond the set temperature for the sample Figure 6 1 When designing transferring to a tube control program it is essential to understand the difference between tube control and block control on a conventional temperature cycling machine With tube ntrol the actual samples are held at the programmed temperature for the programmed time With block control either on the Px2 or a thermal cycler without tube control there will be a lag between the block reaching target temperature and the sample reaching target temperature Thus when transferring protocols from a block control machine the incubation times may be reduced by up to 50 and in some cases the temperatures adjusted slightly For Active Tube Control reactions check that the tube thermistor is connected and located in the block the tube thermistor should be placed in position A2 When you receive your unit the thermistor is mounted in a HBTC3372 tube for 0 2ml blocks or a HBTC3505 tu
56. ivity Stage l Enter parameters to create 95 C 30s 55 C 30s x 14 Cycles 72 C 30s Stage 2 Enter parameters to create 95 C 30s 55 C 30s x 11 Cycles 72 C 30s 5s cycle 1 To enter the 5s cycle select PROG followed by EDIT 2 Inthe EDIT mode NEW or EXISTING move Stage 2 Step 3 relevant extension step using the navigation keys 3 Press ADVANCED to access the Advanced Edit screen Thermo Hybaid Issue 1 0 January 2002 26 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 Using the UP and DOWN softkeys move the cursor through the fields until you reach the desired parameter Use the TOGGLE softkey to alternate between Increase INC and Decrease DEC in set point time cycle 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 Note 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 5 Use ENTER or BACK to get back to the Editing screen lt ADV gt now appears on the screen indicating an Advanced Edit function has been programmed Temperature Advanced Edit Worked Example Temperature decrements can be used for example in touchdown cycling reactions where the annealing temperature is decreased w
57. lculated temperature for each column displayed to the left of the bar 5 Press BACK to return to the Gradient Calculator screen to enter new parameters or alternatively press MENU to return to the Main Menu screen Thermo Hybaid Issue 1 0 January 2002 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Gradient Calculator may be accessed at any time whether the block is active or inactive Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 30 Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 31 CHAPTER 6 Px2 THERMAL CYCLER Running a Program on the Px2 6 1 The Run Option The RUN option in the Main Menu enables you to e Runa previously stored program e Choose the method of temperature control to be used with your protocol e Choose whether to run your samples oil free i e heated lid option Once a program is selected the program name will be displayed and lid preheating if selected will commence It is possible to edit programs whilst the Px2 is running a different protocol see Section 5 3 If a program is selected and an inappropriate temperature control method is used e g tube control or simulated tube control for an in situ interchangeable block module si
58. libration factor of 25 Using a higher calibration factor accelerates the approach to target temperature but results in a degree of overshoot Conversely a lower calibration factor slows the approach to target temperature but minimises any overshoot A calibration factor of zero would be equivalent to block control when using SIM SLIDE Protocol Transfer When transferring protocols from a conventional block control machine it is important to consider the effect of using Px2 s Simulated Slide control On conventional thermal cyclers the programmed temperatures commonly refer to the temperature achieved by the block and not the microscope slide Consequently a sample on the top surface of a glass slide will be significantly cooler than the block temperature The Px2 s Simulated Slide control overcomes this problem by using an algorithm to ensure the temperature achieved by samples on the surface of the slides is as close as possible to the programmed temperature Thermo Hybaid Issue 1 0 January 2002 63 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com In practice this difference in achieved temperature between different control systems is most pronounced at the denaturation step As a guide we recommend that when transferring protocols from a conventional thermal cycler to the Px2 Jn Situ the denaturation temperature is reduced by 3 C Programming Programmin
59. m If complete drying is not allowed the solvent vapour will damage the surface of the Humidity Chamber More information on the use of In Situ applications using Thermo Hybaid equipment can be obtained from the Thermo Hybaid In Situ Hybridization Guide which is available free upon request Thermo Hybaid Issue 1 0 January 2002 64 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com APPENDIX IV Px2 THERMAL CYCLER Glossary of Terms 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 Control The temperature of the cycling block is monitored and controlled by the thermistor mounted on the block Because of the time lag between the block and sample reaching temperature we recommend that tube control is used Block Module The interchangeable assembly comprising sample block Peltier array heatsink and control thermistor Control Chassis The main body of the machine incorporating keypad control and power functions fan and display The Heated Lid is fitted to the control chassis prior to editing programs protocols Software temperatures after gradient cycling 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 u
60. ming Worked Example oornnoronvonnnrnnvrnnvvevenvnnvnnnrn 28 Invalid Gradient Parameters 0 0000 err 29 Gradient Calculator Function 29 CHAPTER 6 RUNNING A PROGRAM ON THE Px2 31 6 1 The Run ONG hvem 31 6 2 Running a Program on the Px2 Short Instructions 31 6 3 Run Screens svurasssndliseivavt rd ak aska na va aaa ga a kala 32 Text Sereenuu sa 32 Power Failure Screen 33 End of Run Screen senravevvenvavevvennnvevsvnnvvevevnnnnevevnnnnuvevnnnnevenannnuseveennee 33 Eror Sere I UR EAN 33 6 4 Temperature Control Options 0 00ee eter 33 Active Tube Control ACTIVE TUBE rorvrrrrrrornnnnnnrrrnnrrrnrnennnnrnnnene 33 Extension Lead for Tube Thermistor 0000 34 Simulated Tube Control SIM TUBE cccccessceesssseeeesssseeeeeeseaes 35 Simulated Plate Control SIM PLATE 00 00000eeee eee eeen 35 Simulated Slide Control SIM SLIDE orvrrrorronnnnnnnrnrnvnrnnrennennnnrnnnen 36 Block Control oraranvwrrrrnnnnnnnnnnvvnnsrnnnnnnnnnnennnsrnnnnnnvnnnnnnnevenennnsnnnennnenee 37 6 5 Heated Lid Preheat eocosasorrrnnnnnnrnnnnnnnnnsnnnnnrnnvnnnnnvsvnsnnnnsnnnnnnnenee 37 Heated Lid Automatic Start 0 eccceseeeccccesseecccescececeseeececeeneess 37 Heated Lid Manual Start oororrrrrnrnnnnnrnnvnnrrvnnnnnnnrnnnnnnevevnnnnsnnnnnnnenee 37 No Heated Lad kassen 38 6 6 Hold and Pause Function
61. mulated slide for an 02 05 module then a warning message will appear and the program will be run under the correct mode of control 6 2 Running a Program on the Px2 Short Instructions Temperature control and heated lid functions are referred to in more detail in section 6 4 1 Select RUN from the MAIN MENU 2 Use the UP and Down softkeys to select the source directory and press ENTER 3 Use the PREVIOUS and NEXT softkeys to select the program and press ENTER or the DOWN softkey 4 Select the heated lid operating method whether the heated lid is on or off during the final hold step loading and end run settings using the PREVIOUS and NEXT softkeys to scroll through options and ENTER to confirm selection Thermo Hybaid Issue 1 0 January 2002 32 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 Select the temperature control mode using the PREVIOUS and NEXT softkeys Pressing ENTER starts the run BACK returns you to the previous screen if necessary 6 3 Run Screens When a program is running two different run screens can be displayed The first screen is the text screen which contains all the information about the progress of the run together with the temperatures achieved during the run This allows the user to monitor the performance of the Px2 and to determine the time of the end of the program The second screen is the graphical display which shows the block a
62. n Press ADVANCED to access the Advanced Edit screen Enter your required gradient range from 1 15 C under GRAD The Program Edit screen will now display the gradient spread entered as lt G 15 gt If a time increment decrement has been activated then lt ADV gt will also be displayed on this screen Proceed though the programming as discussed in section 5 3 Invalid Gradient Parameters If the combination of the Set Annealing Temperature and Gradient results in the block operating outside the temperature limits of 30 to 70 C the Px2 alters the temperatures to remain within these limits E g SET TEMP 60 C and GRAD 15 C The SET TEMP will be changed to 55 C If a temperature is entered that exceeds the 15 C gradient range then a error message will appear beside the GRAD parameter A gradient range from 1 15 C will need to be re entered Gradient Calculator Function Once you have run your samples on an agarose gel the annealing temperature that corresponds to your optimum gel result can be determined using the Gradient Calculator function 1 To access the Gradient Calculator select OPTS from the MAIN MENU 2 Move the cursor to CALC and press ENTER 3 The block type you are using will automatically be displayed Enter the annealing temperature and gradient range used in your optimisation experiment 4 The well temperatures are displayed in sequence in a bar chart formation from column 1 to 12 with the ca
63. n err 14 GlOC Kansas E Ra 14 Power Failure Resume danna 15 4 5 Gradient An 15 4 6 Running a Program eeonvvororrvorrnrnnrnrrnnerrrnsenrrnnsnrrnssnrrsennrsvennrssern 16 4 7 0 0610 0731 102 51 in RA A SRI EIS 16 4 8 Programming Function 00000000ee err 17 4 9 Loading Samples and Heated Lid Operation 18 CHAPTER 5 PROGRAMMING THE Px2 00 000 eeen 19 SI nk 19 Dress 19 Creating a New Del Lavvo 19 Editing a Directory Name ccc ssancesticrsasnsdcdeantndentesstivereombadesnnieanepiwiesrabs 20 Erasing an Existing Directory e euesesariaassesatesstesasnneanensanessvesnarsnnnsnsdeen 20 5 3 PUMPS ecesna a R aS 21 Creating a New PLOStA s 000 a aaa haka aaa a 21 Editing an Existing Favre 22 Viewing an Existing Program ssrovnrnnvnrvonvvnnnvnnnvanenrnsnvnnnnnenrasenrnsnenne 24 Copying eee 24 Erasing an Existing Program s4iomsmikeeimmensmmssietemeadentetddnednedede 25 5 4 Advanced Edit Features uuoosnumsasmmenagnnseetdetekimne 25 Thermo Hybaid Issue 1 0 January 2002 vii Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Time Advanced Worked Example ceccesseessecsseceseeeeeeesseeeseeeeees 26 Temperature Advanced Worked Example sonnrrnnnvoronvvnnnrnvvrnnvvrvrnnnnr 27 Changing Ramp Rates unmmniggemnnsanneitedtjuinumeu dm 28 5 5 Gradient Feature 28 Gradient Program
64. nd NEXT keys to change to the appropriate method A BACK button can be used to return to the previous screen if necessary Pressing ENTER will now start the program Prior to starting the program pressing the MENU key at any stage will return to the Main Menu More details can be found in Chapter 6 4 7 Manual Operation This option is used for static incubations when thermal cycling is not required e g probe denaturations and enzyme reactions In this mode the unit will only operate under block control see Chapter 6 for details 1 Select MAN from the Main Menu by depressing the relevant softkey 2 Enter the name of the user or protocol if desired using the alphanumeric keys Move to the next previous character using the LEFT and RIGHT arrow softkeys Pressing ENTER moves the cursor to the temperature selection this occurs even if a user name has not been entered 3 Enter the temperature using the alphanumeric keys Press ENTER to continue 4 Select the heated lid on auto or off using the LEFT and RIGHT softkeys Thermo Hybaid Issue 1 0 January 2002 16 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com NB 4 8 Press ENTER to start the incubation The screen displays the target temperature and actual temperature of the block together with the elapsed time The count up timer will start when the block reaches temperature If the machine is started fr
65. nd tube sim tube temperatures The screens may be toggled between using the LEFT softkey TEXT GRAPH These screens can be accessed from the Main Menu by pressing the RUN INFO softkey Text Screen The current block performance screen displays the following e Program number and name e Status of the program e The temperature control mode being used e Target programmed temperature for a given step e The block and tube temperatures e Time remaining at current setpoint e The current stage and step of the running program e The total number of cycles completed and remaining e The maximum and minimum temperatures achieved during the run in the respective control mode e Time now e The calculated run end time an estimated value which is updated throughout the run During a gradient step the run screen also displays the following e Target temperature range of the set gradient Thermo Hybaid Issue 1 0 January 2002 33 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com e Temperature range achieved e The actual sample temperature as defined by the tube thermistor in position A2 of the block when Active Tube Control has been selected for a run Power Failure Screen This additional screen appears if the run has been interrupted by a power failure or power fluctuation severe enough to affect the unit It contains the following information e The duration
66. of the power failure e The stage in the run when the power failure occurred End of Run Screen The Px2 displays a further screen at the end of the run which provides the following e The program name and number e The total run time e The maximum and minimum temperatures recorded during the run e Power interrupt information if a power failure occurred Error Screens A number of error screens may be displayed if a fault has been detected in the unit Contact the Thermo Hybaid Service Department or your local supplier for advice before attempting to use the instrument further 6 4 Temperature Control Options Crucial to the accurate operation of a thermal cycler is an understanding of the temperature control methodology Active Tube Control ACTIVE TUBE Recommended for all reactions above 20ul volume in 0 5ml and 0 2ml tubes This type of control uses the remote thermistor probe mounted in an appropriate tube A volume of mineral oil equivalent to the total volume in the reaction tubes including any oil overlay must be present in the control tube Thermo Hybaid Issue 1 0 January 2002 34 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note Please do not use aqueous solutions in the control tube thermistor We recommend that mineral oil be used in the tube containing the thermistor because the software is tuned to take into consideration the thermal conductiv
67. om 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 tempe rature Press the STOP softkey followed by ABORT to cancel the program Press the NEW TEMP softkey to change the set temperature and then CONTINUE to continue the program with a new temperature Programming Function PCR protocols of varying degrees of complexity can easily be created using Px2 As with all operations of the Px2 programming is accessed from the Main Menu Access the Program Menu by selecting PROG from the main menu If at any time you wish to stop programming press MENU to return to the Main Menu Four options are available for programming The LEFT or RIGHT arrow keys are used to move the cursor to the required menu option Press ENTER to then select that option 1 EDIT To alter the details of an EXISTING program or to create a completely NEW program or directory Note that if NEW is used it will automatically move to the next available program number COPY Use the screen prompts to copy between programs stored on the Px2 VIEW To check the details of a previously stored program ERASE To erase the details of a program setting the parameters to zero These are discussed in detail in Chapter 5 Thermo Hybaid
68. polycarbonate plates supplied by other manufacturers a minimum sample volume of 25ul should be observed to ensure adequate sample remains after PCR Thermo Hybaid Issue 1 0 January 2002 62 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com APPENDIX IIT Px2 THERMAL CYCLER In Situ Block Application Notes Overview The Px2 Jn Situ module with flat block has been designed for the precise control of temperature on up to four standard microscope slides dimensions 76mm x 26mm x 1mm Flat blocks are supplied with a Humidity Chamber which can be used to reduce sample drying Temperature Control The In Situ block can perform all the control functions of the standard Px2 reaction tube block with the exceptions of Active Tube Control and Simulated Tube Control Instead the Simulated Slide Control option is used which minimises the time lag between the block reaching target temperature and the sample on the surface of the slide achieving target temperature At the start of running a program on an In Situ module the SIM SLIDE option should be selected as the control mode see Figure 5 1 The heated lid is not operational under this control mode The performance of the In Situ module has been measured using miniature thermistor probes attached to the surface of slides Optimum results across four standard slides Imm thickness with the humidity chamber in place have been obtained using a ca
69. possibly the heater plate of the heated lid 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 minimise contamination 1 Use a mineral oil overlay in all reactions even when the heated lid is used 2 If using tubes use only the thick walled variety 3 Use the thermal cycler in a fume hood to minimise air contamination 4 If using microtitre plates coat the under surface with a thin layer of mineral oil 5 If radioactivity must be used the thermal cycling block and heated lid surfaces can be decontaminated using a 10 v v solution of Neutracon Decon Laboratories Ltd Conway Thermo Hybaid Issue 1 0 January 2002 47 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Street Hove East Sussex BN3 3LY Tel 44 0 1273 739 241 Fax 44 0 1273 722 088 or PCC 54 Pierce Chemical Company 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 Control Chassis Block Module and Heated Lid 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 Hybaid s Service Department so that the appropriate handling arrangements can be mad
70. program choices MENU KEY Always moves to the Main Menu can be utilised as an escape key ALPHANUMERIC Used for entering parameters KEYS Used for selecting programs Alphanumeric naming of programs directories SOFTKEYS These keys alter function depending on the screen and the state of the block e g active or inactive Start Stop programs Navigate around the screen Navigate between screens Used to toggle between options 4 4 Set Up Functions Selecting OPTS at the Main Menu by pressing the relevant soft key gives access to the SETUP menu Within the SETUP screen information about the type of block fitted the current software version installed the time and the date are displayed A flashing cursor beneath CLOCK now indicates that this application will be selected when ENTER is pressed The LEFT or RIGHT arrow keys can be used to alter the selection and a BACK button is available to return to the previous screen Clock The time and date format of the clock display can be modified To set the clock or alter the format select the SETUP screen followed by CLOCK The American MM DD YY or European DD MM YY format can be selected using the TOGGLE softkey Thermo Hybaid Issue 1 0 January 2002 14 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Press ENTER or use the PREVIOUS and NEXT softkeys to move to the time date setting fields Use the number ke
71. rally in the control tube and immersed in the liquid If pushed against the side it will be measuring the temperature of the microcentrifuge tube and not the sample temperature Check the Fit of the Tube in the Block The Px2 block accommodates most types of reaction tubes The tube thermistor supplied with the Px2 is mounted on an OmniTube which should be changed periodically as the fit of the tube will deteriorate with time Reaction tubes should be distributed evenly in the block If you are using a reaction tube that has not been recommended check its fit in the Px2 block before use You should also remount the thermistor probe in this tube to ensure that your samples and the control tube are matched Check through the Program Before running a program use the VIEW function to check that the correct combination of temperature and program number have been entered Thermo Hybaid Issue 1 0 January 2002 42 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 7 2 Programs Using SIM Temperature Control Simulated Tube Enter the number of samples including the thermistor and the total reaction volume per tube including any oil overlay 50ul should be added to the volume factor if thick walled tubes are used Use a setting of 96 samples and the correct reaction volume including oil overlay if a polypropylene OmniTube 96 is used Simulated Plate 0 2ml blocks For 96 well mic
72. roplates including OmniUltra 96 on a 0 2ml block the volume factor entered is the individual sample volume Note Polypropylene Omni Tube 96 should be run under Sim Tube Control 0 5ml blocks For 0 2ml OmniPlates on a 0 5ml block the volume factor entered is the total sample volume multiplied by 10 to a maximum of 500 This accommodates the non optimal fit of the OmniPlate 96 in the 0 5ml block Other 96 well plates for example the Costar ThermowellH may be used with lower multiples of volume but these need to be individually determined 384 well blocks For 384 well blocks the individual well volume should be used for the volume factor Simulated Slide For 0 8mm Imm thick glass slides using simulated slide control the calibration factor is 25 default value other thicknesses will need optimisation Check maximum and minimum values achieved during the run This will give an indication of unusual temperature performance Power failure during a run A 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 specified by the user on the SET UP menu screen Viewing the run screens for the block will indicate the time of the power failure and where applicable the time the run resumed Thermo Hybaid Issue 1 0 January 2002 43 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com
73. rumentation Guaranteed 888 88 SOURCE www artisantg com 9 Enter any changed cycle number or hold temperature values Press ENTER to proceed to the next stage 10 Edit steps in further stages as required Once changes are completed the SAVE button can be pressed to access the save screen 11 Press the SAVE softkey to save changes or IGNORE to keep the original program Pressing MENU at anytime aborts the editing Viewing an Existing Program It is possible to view existing programs without altering the information contained within them 1 Select PROG from the Main Menu and then use the RIGHT arrow keys to move the cursor to VIEW from the program menu Press ENTER to select 2 Use the UP and DOWN softkeys to select the appropriate directory and then press ENTER 3 Use the PREVIOUS and NEXT softkeys to select the program and press ENTER 4 Use the NEXT softkey to scroll through the program information Press QUIT or MENU at any time to return to the Main Menu Copying Programs Copying programs is necessary in for example modifying the pre set programs described in Appendix V 1 Select PROG from the Main Menu and then use the RIGHT arrow keys to move the cursor to COPY from the program menu Press ENTER to select 2 Move the cursor using the UP and DOWN softkeys to select the source directory and press ENTER 3 Choose the program to be copied by using the PREVIOUS and NEXT softkeys and press ENTER to select
74. rumentation Guaranteed 888 88 SOURCE www artisantg com is as close a representation as possible to the biological samples To maintain accuracy and longevity of the tube thermistor Thermo Hybaid recommends the following 1 Do change the tube thermistor annually It may be necessary to change the thermistor more often under heavy use e g frequent removal of the thermistor from the tube 2 Do check that the mineral oil volume is the same as the reaction volume including any oil overlay 3 Do check and replace the control tube if it is showing signs of thermal degradation 4 Do use the same type of tube for the thermistor tube as for the biological samples 5 Do make sure the sensor part of the thermistor probe is centrally located at the base of the tube and is fully covered by mineral oil 6 Don t use aqueous solutions in the thermistor tube 7 Don t remove the control tube from the cap assembly by pulling the wire gently lever the tube from the cap 8 Don t kink the thermistor wire or crush the sensor part of the thermistor probe end 9 Don t remove the thermistor assembly from the instrument by pulling on the wire pull from the plug Thermo Hybaid Issue 1 0 January 2002 49 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Thermo Hybaid Issue 1 0 January 2002 Artisan Technology Group Quality Instrumentation Guaranteed 888 88
75. sed to hold samples at a fixed temperature while a reagent is added before temperature cycling starts four microscope slides number of tubes for each cycling run MAN Manual operation is used for single temperature incubations Manual Operation under block control The incubation will proceed at the set temperature until the STOP button is pressed or until a new temperature is specified The menu key allows the user to return to the main menu at any time without making changes in the programming operation of the unit CALCULATOR Software Thermo Hybaid Issue 1 0 January 2002 65 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com New Temperature A 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 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 Failure The Px2 has the facility to restart after a mains power supply failure There are two set up options for restarting after a power failure 1 Resume program at step that power failure occurred 2 Abandon program at power failure step One of the above should be specified using the SETUP menu
76. te the tube in a well of the block control machine running the required protocol 4 After a short equilibration interval the display on the Px2 will indicate the sample tube temperature which should be noted at regular time intervals e g 10 seconds for a number of cycles The actual length of time spent at each of the denaturation annealing and elongation stages should be recorded as well as any temperature overshoot values where the maximum minimum temperature exceeds the target temperature or undershoots where the target temperature is not actually reached 5 The temperature profile that the samples in the block control machine actually achieve rather than simply the block temperature can be used to program your Px2 Doing this will significantly reduce the total time required to run a protocol 7 4 Optimisation of Protocols The capability of the Px2 programming enables protocol optimisations to be performed very rapidly Typically temperature cycling protocols may consist of three distinct stages Thermo Hybaid Issue 1 0 January 2002 44 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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
77. 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 Thermo Hybaid Issue 1 0 January 2002 4
78. three steps are typically repeated for twenty to thirty cycles depending on the amount of starting template Denaturation The denaturation step at each cycle must be sufficient to denature the target DNA completely including GC 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 Optimisation of the denaturation step is the most critical factor when transferring a protocol from a block control machine to tube control 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 2x A T 4x G 0 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 optimise the annealing temperature for each primer and template combination This is best performed using the gradient feature described is section 5 5 Extension The extension temperature is largely dependent upon the optimum temperature of the enzyme chosen and is usually
79. to 5 nuclease assays or to any patent claiming a reagent or kit Applied Biosystems does not guarantee the performance of this instrument Thermo Hybaid Issue 1 0 January 2002 iii Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chassis Heated Lid Height Selector Heated Lid Large LCD Screen Key Pad Main Body Stabilising Feet Thermo Hybaid Issue 1 0 January 2002 iv Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 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 th
80. tubes with integral flat caps HB TC 6202N OmniTube 0 5ml 1000 0 5ml individual tubes with integral domed caps HB TC 4895N OmniTube 0 5ml 1000 0 5ml individual tubes with integral flat caps HB TC 3505N OmniStrip 0 2ml 2000 0 2ml strips of 8 tubes amp caps HB TC 2662N OmniStrip 0 3ml 2000 0 3ml strips of 8 tubes amp caps HB TC 4043N Racked OmniTubes For 0 3ml 96 well plates caps not included HB TC 4073N OmniTube Caps 2400 For all 0 2ml and 0 3ml OmniTube products HB TC 6022N We recommend that domed cap tubes are used when the temperature control is set to Active Tube and flat cap tubes are used under Simulated Tube Control Polycarbonate can be affected by contact with formamide DMSO and other organic solvents these should therefore be avoided when using the polycarbonate OmniUltra Plate Thermo Hybaid Issue 1 0 January 2002 58 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com APPENDIX II Px2 THERMAL CYCLER Sealing Systems The OmniSeal TD Mat amp OptiTape Overview Thermo Hybaid offers several sealing systems to suit individual preferences OptiTape is an optically clear single use adhesive tape suitable for use with 96 well polycarbonate plates whilst the OmniSeal TD Mat is a reusable silicone rubber mat also for use with 96 well plates For polypropylene plates we supply adhesive TDX Tape How to use the OptiTape and TDX Tape OptiTape and TDX T
81. vanced Edit Gradient optional eoonrrrnrvrrronvvnrnvnenrnvennnrnvnnnnnnenn 4 Calibration EE 4 1 3 The Heated Lid ju nvmigsmnsmimsiensredaasieviasarpdetde 4 Operating the Lil 230 a a aa a la a la idea ai a 4 14 Input Output Sockets isss skan snu ks 6 1 5 Support Sel 6 CHAPTER 2 SAFETY PRECAUTIONS rrrornrnvrrronrrennnnrnrnrrrrvennesnnnnerrnrerseennessnnnerrseenne 7 2 1 General Safety Precautions rrrrrnnrrrnnvvrrnnrvrrnnvrrrnnvrrrnnvrrnnnvnrnnnnnn 7 2 2 Symbols and Conventions cccceeceeseceteceseeeeeeeseeenaeenseensees 8 CHAPTER 3 UNPACKING amp INSTALLATION 0 0000 eeen 9 M Unpacking ieena a a baea n i aea E Ria n 9 3 2 GE RE ET aan 9 Thermo Hybaid Issue 1 0 January 2002 vii Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 3 Installation err 10 EGO sia saka jeni ga Ain al ep Eai 10 Block Installation and Removal 00eeeeeeereer erase 10 Thermistor POSitioning 4 sicasisassacsassnscevastdeadesadvescsasnacensasbevasssseesnesanbepeeds 11 sem Cold ROOMS eirinn rsa aS 11 CHAPTER 4 OPERATING INSTRUCTIONS rrrarrrrrvrrrronvrrnnrrrrrrrrrrsnvrrrnnrrrrsnvnrrnnnne 13 4 1 Initial Start Up vcs ccs ieee ee ee es 13 22 Main Men ast n a a a 13 4 3 Operating Keys sasssa s nd a i a 14 4 4 Set Up Functions eee eee
82. 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 When using a0 5ml block 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 QO m Figure 4 1 Open Lid Icon Tube Icon Plate Icon Thermo Hybaid Issue 1 0 January 2002 18 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com CHAPTER 5 Px2 THERMAL CYCLER Programming the Px2 5 1 Introduction The Px2 has been designed to make programming user friendly and easy The memory will hold up to 99 complete thermal cycling protocols including 10 pre programmed template programs Nos 90 99 detailed in Appendix V Programs are identified by a number 01 99 and a user defined name up to 7 characters Each program protocol type may be assigned to a directory enabling you to group programs by user name To enter a new program you simply 1 Enter or create a new directory 1 Name a program iii Enter protocol parameters iv Save the program 5 2 Directories There are five directories A E which can be individuall
83. y named The sixth directory F is reserved for pre set programs Directories can also be erased and 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 Creating a New Directory 1 From the Main Menu select PROG using the appropriate softkey 2 With the cursor highlighting EDIT press ENTER 3 Use the LEFT and RIGHT navigation key to highlight NEW with the cursor and press ENTER Thermo Hybaid Issue 1 0 January 2002 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Use the LEFT and RIGHT navigation key to highlight NEW DIR with the cursor and press ENTER Select the desired target directory A E using the UP and DOWN softkeys and then ENTER You are now able to name the directory using the alphanumeric keypad Use the PREVIOUS and NEXT softkeys to move to the position of the cursor where the character is to be added Press ENTER only when the name is complete If no characters are entered the directory name will remain blank The BACK button may be used to return to the previous screen if necessary Editing a Directory Name To change the name of an existing directory simply retype the new directory name over the old one Select PROG from the Main Menu using the appropriate softkey The cursor should be flashing below EDIT select this option by pressing ENTER
84. ys to enter time hrs amp mins and day month year information Press BACK to the previous screen or ENTER to confirm changes and return to the Main Menu Power Failure Resume If there is a power interruption during a run the Px2 will switch back on automatically The unit can be set to resume a program at the cycle and setpoint where the interruption occurred or to abandon the program Select POWER from the SETUP menu and press ENTER The ABANDON or RESUME options can be selected using the TOGGLE softkey Press the CONTINUE softkey to return to the Main Menu The instrument will record the duration of the interruption and the step stage and cycle number at which it occurred Note that if there have been several breaks in the power supply only the most recent interruption is recorded 4 5 Gradient Calculation Once you have run your samples on an agarose gel the annealing temperature that corresponds to your optimum gel result can be determined using the Gradient Calculator function e To access the Gradient Calculator select OPTS from the MAIN MENU e Using the left and right navigation keys move the cursor to CALC and press ENTER e The block type you are using will automatically be displayed Enter the minimum annealing temperature and gradient range used in your optimisation experiment using the alphanumeric keypad e The well temperatures are displayed in sequence in a bar chart formation from column I to 12

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