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1. Move the cursor Hit the CLICK button Is the touch pad mouse selection switch in the appropriate position Rear position for touch pad function or forward position for mouse function Does the external mouse port function sen Front end OK proceed 10 9 M DNA Engine Dyad Operations Manual Problems with an Alpha Unit An Alpha unit is a distinct separately engineered piece of equipment that is made to inter face with all MJ Research DNA Engine line thermal cyclers However to rule out problems with any Alpha unit please follow this recommended troubleshooting flowchart Figure 10 3 Alpha Unit Troubleshooting Flowchart System status at this point Front end OK front end OK communicating w engines proceed Problem protocols do not appear to be running properly Check error log Is problem Alpha unit related Alpha unit defective return for service Base defective return for service Problems Related to Protocols The suggestions we make here are by no means exhaustive but are intended as a starting point for further investigation Should the Dyad system check out OK we recommend posi tive controls for troubleshooting purposes Following is a general description of some common problems related to the protocols and reaction components in sequencing and thermal cycling applications For a more detailed discussion of protocol
2. 735 8437 for additional information on the Alpha units listed here as well as updates on any new Alpha units available m DNA Engine Dyad Operations Manual About the Remote Alpha Dock System The Remote Alpha Dock system is designed to add flexibility to the installation and opera tion of the MJ Research PTC 220 DNA Engine Dyad cycler The system allows Alpha units to be placed at a distance from the PTC 220 base enabling more efficient use of space and facilitating robotic operation The basic system the RAD 200 comprises a dock connector which mounts in the cycler base and a Remote Alpha Dock base into which the Alpha units are mounted Additionally the fan power supply along with the cables to run up to four Remote Alpha Dock fans from a single thermal cycler base must be purchased separately as RPS 0200 Figure 11 1 The Remote Alpha Dock base with Alpha unit mounted Alpha Units and the Remote Alpha Dock System E Packing checklist One dock connector One Remote Alpha Dock chassis One multi pin power cable One multi pin data cable One fan power supply RPS O200 One wall plug power cord RPS O200 Three round jack power cords RPS 0200 Product registration card US and Canada only Extended warranty application US and Canada only Requirements Environment The Remote Alpha Dock system allows for custom installations The following placement configurations are recommended by MJ Research Inc Figu
3. A small amount of oil is added to the probe tube to serve as the representative sample The tube is loaded into the block where it can serve as the control reference for any programmed target temperature between 0 and 100 C When a probe control protocol is run the DNA Engine Dyad cycler controls block tempera ture to keep the probe vessel at the programmed temperature using feedback information from the thermistor See Chapter 5 for information on programming protocols for probe control Probe control cannot be used with heated lid protocols Note Because the thermal characteristics of a probe never precisely match those of an actual sample calculated control is often a better choice than probe control Customizing the Probe Vessel For the most precise control of sample temperatures install the probe s thermistor in the same type of tube that the samples will be placed in This is particularly important when the sample tubes have much thicker walls than the probe s tube Follow these steps to customize the probe vessel 1 Cut the hinge to the probe tube s lid if there is one Remove the lid and the attached amber colored thermistor 2 Remove the lid from the new probe tube Add oil to the probe tube as described below under Adding the Oil Figure 4 3 A Probe B Location of probe jack N N Probe tube Probe plug m DNA Engine Dyad Operations Manual 3 Gently place the thermistor in the new tube an
4. Assume you have the necessary components for a 30 cycle sequencing reaction and you have calculated the annealing temperature of your oligonucleotide to be 60 C Please note that MJ Research recommends using 92 C as the default denaturation temperature during cycling steps The resulting raw program you write may look something like this Raw program 1 OF oe ee 92 C for 30 seconds 60 C for 3 minutes 92 C for 30 seconds 60 C for 3 minutes 92 C for 30 seconds 60 C for 3 minutes continues for a total of 60 lines The Goto Option At 60 lines our program is large unwieldy and would take time to input At step 3 repetition can be reduced with the addition of a goto statement Raw program l 2 de 92 C for 30 seconds 60 C for 3 minutes Goto step 1 29 more times END One of the most important factors in the program writing process is identifying repetitive steps These can then be enclosed in a goto loop as shown above Creating Programs W Considerations During Program Creation Once you have written the body of your raw program there are decisions to make before creating your Dyad program They concern how your steps should be implemented These decisions involve the following Temperature control mode Lid control mode Temperature ramping rate advanced mode only Temperature hold time Temperature time extend advanced mode only Temperature increment advanced mode o
5. Call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada This sample block reached a higher temperature than was expected An irregularity was detected in the block s center temperature sensor The sensor will be checked automatically during No action required at this time and alter the run and you will be notified if any persistent oroblem is found An irregularity was detected in the block s left temperature sensor The sensor will be checked automatically during No action required at this time and after the run and you will be notified if any persistent oroblem is found An irregularity was detected in the sensitivity of the block s right temperature sensor The sensor will be checked automatically No action required at this time during and after the run and you will be notified if any persistent problem is found An irregularity was detected in one or more of the block s temperature sensors The Call 888 MJ CYCLE in the US sensor was checked and the or Canada or your local results show an inaccuracy in distributor outside the US or block temperature measurement Canada Your program was automatically stopped L e Heated 1a is nok pre Recover samples and call 888 eating correctly Your samples yy CYCLE lin the US or have remained i roS Canada or your local distributor temperature and your program outside the US or Canada was never
6. Creating Programs W The Program Display box will list steps for the program currently running on the selected block The menu bar at the top of the Status window includes five submenus lt Programs gt lt Com mand gt lt Tools gt lt View gt and lt Utilities gt These submenus provide the operator with paths for maneuvering through the various Dyad software windows For the purposes of this chap ter we will be primarily concerned with the lt Programs gt submenu The other submenus will be covered in Chapters 7 and 8 Entering a Program Using Graphical Mode After creation of the initial program entering a program in graphical mode essentially in volves editing the graphically displayed TEMPLATE program The TEMPLATE program will be the last graphical program that was saved In this section we will address both creating an initial graphical program and editing a preexisting template e Select lt Programs gt Note As described earlier this involves positioning the screen cursor over lt Pro grams gt with a fingertip on the touch pad and tapping the touch pad once Drop down submenus appear including lt Open gt lt New gt lt Copy gt lt Move gt lt Delete gt lt Delete Folder gt and lt New Folder gt e Select lt New gt An additional menu appears allowing you to choose lt Advanced Mode gt or lt Ba sic Mode gt e Select lt Basic Mode gt Program TEMILATE E Lid Mode CONSTANT 100
7. FCC Warning Changes or modifications to the the PTC 220 DNA Engine Dyad thermal cycler not ex pressly approved by the party responsible for compliance could void the user s authority to operate the equipment Appendix B How a Peltier Heat Pump W orks W Appendix B How a Peltier Heat Pump Works The functional heart of every DNA Engine Dyad cycler is a high performance Peltier effect heat pump also known as a thermoelectric module The MJ module is a solid state device manu factured to withstand the thermal stresses associated with rapidly cycling temperatures A thermoelectric module consists of numerous pairs of crystalline semiconductor blocks precisely sandwiched between two layers of ceramic substrate figure A 1 The blocks are of two variet ies N type which has a surplus of electrons in its crystalline structure and P type which has a deficit of electrons The two types are posi tioned in alternating pairs within the innermost layer of the sandwich Figure A 1 A thermoelectric module The two types of blocks are wired together in alter A tC nating pairs When electrical current is passed So Roue the blocks electrons in the N type blocks DOO AT Cy and the holes or empty electron spaces in the P Power input A Ceramic substrate type blocks are excited at one conductor semicon Oj Metal conductor ductor interface which absorbs a small amount of heat The electrons and holes flow
8. Lid Control Mode The Hot Bonnet heated lid can be programmed to minimize condensation by keeping the upper surface of the reaction vessel at a temperature slightly greater than that of the sample itself The three available lid modes include Off Tracking and Constant The Dyad cycler defaults to Constant Refer to the Choosing a Lid Control Mode section below for additional information Creating Programs W Temperature step This sets incubation temperature and duration The Dyad cy cler ramps the sample to this temperature at its maximum rate unless ramp modifying instructions are added to the program advanced mode only The maximum rate of heating is 3 C sec and cooling is 2 C sec for all standard Alpha units maximum rate of heating is 1 2 C sec for the Twin Towers Alpha unit Gradient step This establishes a temperature gradient across a 96 well sample block The range of any single gradient can be as great as 24 C or as small as 1 C trom left to right across the block The maximum programmable temperature is 105 C the minimum programmable temperature is 30 C GoTo step Directs the program to cycle back to an earlier step a specified number of times Lid step Directs a Power Bonnet motorized lid to automatically open or close only available in advanced programs End step Automatically included this instructs the Dyad cycler to shut down its heat pump because the program is complete These additional pro
9. US Underwriters Laboratories UL Std No 3101 1 Electrical Equipment for Laboratory Use Part CSA Int Certificate 1125786 LR 97357 10 US This device complies with Part 15 of the FCC Rules voluntarily TYPE OF EQUIPMENT European Union Class A Electrical Equipment for Measurement Control and Laboratory Use Mobet NUMBER Model PTC 220 DNA Engine Dyad thermal cycler rated 200 240VAC 50 60Hz 1 6 kW cord connected CSA International certifies as Equipment Class Pollution Degree 2 Installation Category II YEARS OF MANUFACTURE July 2001 and onward MJ RESEARCH INCORPORATED manufacturer of the equipment described above certifies this model of instrument has been tested and conforms to the applicable Directives amp Standards of the European Union EU as well as those for Canadian and US compliance as described above Test data to verify this conformity are available for inspection at MJ RESEARCH INCORPORATED both at the U S manufactory detailed below as well as at the MJ Research European Representative Office at Kirke Vzerlosevej 16 3500 V rl se Denmark 45 44 35 05 40 fax 45 44 35 05 49 BE oP ef DZ Soe Robert A lovanni ate signed Electrical Engineer on behalf of MJ RESEARCH INCORPORATED 590 Lincoln Street Waltham Massachusetts 02451 USA tel 617 923 8000 fax 617 923 8080 Copy number ORDERS via phone 888 729 2165 via fax 888 729 2166 via e mail orders mjr c
10. e Connect the multi pin power cable s female end to the connector labeled Power Port and slide the latch to lock the pins in place e Attach the data cable s DOCK DATA end to the connector labeled Data Port and slide the latch to lock the pins in place e Turn both units back over e The dock connector mounts in the base in the same manner as a regular Alpha unit see Operating Alpha Units in Chapter 4 e Attach the wall plug power cord to the fan power supply e Attach the fan power supply s round jack to either of the round connectors on the back of the Remote Dock fig 1 1 4 e The fan power supply produces sufficient current such that up to three additional fans can be daisy chained to the initial Remote Alpha Dock unit via the round jack power cords supplied Attach one end of the cord to the initial unit s free round connector Attach the other end to either of the round connectors on the back of the next Remote Alpha Dock unit in the series and so on fig 1 1 4 e When the Remote Alpha Dock system has been completely set up connect the fan power supply s wall plug to a power source Operation The Remote Alpha Dock system is transparent to the base unit i e the dock connector allows the base to control the Alpha unit in the Remote Alpha Dock base as if it were in the standard configuration 1 IMPORTANT Turn the base unit s power off when changing the type of sample block you a
11. is no Power Bonnet lid installed Lid mode Alpha units will be grayed out if the lid mode is not set to OFF when using a block with no lid such as a Twin Towers Alpha unit When encountering an inaccessible feature or block please review your program with the installed Alpha units to determine if an incompatibility is present Running Multiple Programs One particularly useful feature of the Dyad cycler is the ability to run several programs at once on different blocks For example in a Dyad cycler setup with two 96 well Alpha units a gradient for optimizing annealing temperature can be run in one Alpha unit whereas a typical experiment without a gradient can be run in the other Alpha unit simultaneously Before running multiple protocols considerations should be made as to the compatibility of the program with the available Alpha units Please review Chapter 4 Operation for a more complete treatment of Alpha units Running Programs E To run multiple programs first choose an available block from the block selection menu in the Status window Available blocks will show a Block is Inactive message just above the User Name field If there are no available blocks the No blocks available message window appears Select lt Run gt and follow the instructions in the Running Protocols section in this chapter fo initiate an independent run To summarize we have learned how to run programs on the Dyad cycler We hav
12. liquid wax or the probe readings will not be accurate Caution Do not use water saline or any other aqueous solution as a representa tive sample Aqueous solutions will destroy the thermistor Loading and Connecting the Probe Seat the probe tube in the center of the block see figure 4 4 If oil is used to thermally couple samples to the block it must also be used on the probe tube see Using Oil to Thermally Couple Sample Vessels to the Block Plug the probe into the jack at the back of the block so that the wire is to the left of the plug see figure 4 4 Operation W Detecting a Faulty Probe If the DNA Engine Dyad cycler detects that the probe is broken or missing when a protocol begins running the protocol s temperature control method is automatically switched from probe control to calculated control and the following message is immediately displayed Probe Sensor Failure Used Calc Control If the probe malfunctions during a protocol run the temperature control method is also switched to calculated control When the run finishes the following message is displayed Calc control Probe not present Figure 4 4 Correctly inserted probe Probe wire exiting Probe plug seated to left of plug In jac 4 13 m DNA Engine Dyad Operations Manual Appendix 4 A Tube Microplate and Sealing System Selection Chart The following sample vessels and sealing options are rec
13. signed to ensure that programs are appropriately saved Select lt Save As gt 5 17 M DNA Engine Dyad Operations Manual 5 18 File Save As x Hew Folder Program Name TEMPLATE Edit Name DE Cancel SAMPLE The File Save As window presents the operator with a space for entering the program name The program will be added to the folder indicated in the Folder tield The lt New Folder gt button creates a new folder in which to store your new program Select lt New Folder gt Edit ReyPad ijeja s z gt a 1 2 3 4 5 6 7 8 s9 0 lt In this Edit KeyPad window you can select letters that will compose the name of your new folder Folder names cannot be longer than eight characters The virtual keyboard will be presented in situations where a combination of let ters and numbers should be entered Select the characters F O L D E R 1 in succession The backspace key can be used to correct any mistakes Select lt OK gt Creating Programs W You will be returned to the File Save As window The display will have changed slightly with our newly created folder appearing in the Folder list We will want to save our program in this newly created folder e Position the cursor over the folder FOLDER1 and select e Select lt Edit Name gt Again you are presented with the Edit KeyPad window e Select the characters G R A P H 1 in succession e Select lt OK gt Yo
14. 0c 55 0 55 oO 30 00 30 OO 30 Add Step after Control Mode catc Grad Golo Forever Delete Step Save Save As Save Hun Cancel The graphical programming window appears displaying no program steps or the last saved program In either case the new program bears the default name TEMPLATE 5 15 M DNA Engine Dyad Operations Manual Begin by choosing the temperature control mode and lid control mode for the program Refer to the Considerations During Program Creation section earlier in this chapter for information on temperature and lid control modes The current mode of temperature control is listed in the Control Mode field The current mode of lid control is listed in the Lid Mode field To change the control or lid mode select the box in front of that field In either case the Mode Selection window appears Using the Mode Selection Window Hode Selection x Temperature Control Mode T Block in Sample Probe Lid Control Mode W Constant Tracking Set Parameters Off OF Cancel For the purposes of this example we have decided to use Calculated for our Temperature Control Mode e Select Calculated We have decided to use Constant for our Lid Control Mode e Select Constant Constant mode will allow the operator to set the parameters for the heated lid temperature as well as the temperature at which the lid will turn off e Select lt Set Parameters gt The
15. 6 Installation Packing Checklist 3 2 Setting Up the DNA Engine Dyad Cycler 3 2 Optional External Mouse Device 3 2 Environmental Requirements 3 3 Power Supply Requirements 3 3 Air Supply Requirements 3 4 Ensuring an Adequate Air Supply 3 4 Ensuring That Air Is Cool Enough 3 4 Requirements for Robotics Installations 3 5 384 Well Microplate Specifics 3 6 E DNA Engine Dyad Operations Manual 3 2 Packing Checklist After unpacking the DNA Engine Dyad cycler check to see that you have received the following One DNA Engine Dyad base Two spare fuses One power cord One external mouse device PTC 220 DNA Engine Dyad Peltier Thermal Cycler Operations Manual this document Product registration card US amp Canada only Extended warranty application US amp Canada only If any of these components are missing or damaged contact MJ Research or the authorized distributor from whom you purchased the DNA Engine Dyad cycler to obtain a replacement Please save the original packing materials in case you need to return the Dyad cycler for service See Appendix C for shipping instructions Setting Up the DNA Engine Dyad Cycler The DNA Engine Dyad cycler requires only minimal assembly plugging in the power cord and mounting the Alpha units Insert the power cord plug into its jack at the back of the machine see figure 2 3 for location of jack then plug the cord into a 220V electrical outle
16. Auto Remote option The cycler will now power up in standard operation mode Using the Utilities Hi lt Update Soft gt amp lt Network Config gt These utilities allow you to update both the software controlling the Dyad cycler s hardware i e engine software and the user interface programming software i e front end soft ware by establishing an internet connection and directly downloading any new software upgrades or versions If you are unsure of the current version of engine and or front end software installed on your Dyad cycler use the About utility as described in the beginning of this chapter to view the current software versions Front end software version 1 07 or greater is required to use the lt Update Soft gt and lt Network Config gt utilities To view the available software upgrades and or perform an upgrade begin by establishing internet access via an Ethernet 10BaseT connection Connect the ethernet cable to the ethernet port located at the rear of the Dyad cycler see figure 2 3 Then select lt Update Soft gt from the lt Utilities gt menu HTTP Update Software Utility Select Software Yersion Front End Version 1 07 Dec 21 2001 ya Display Versions Update Log Hetwork Config The HTTP Update Software Utility window appears Begin by specifying the type of connec tion that you have to the internet by selecting lt Network Config gt The FTP Server Configura tion window appears Yo
17. C Step 00 00 00 Sample 248 C Lid 23 4 C Cycle 0 Aun Instant SIP Pause GRIP Graphs Remaining 00 00 00 e Select lt Instant gt at the bottom of the window or from the lt Com mand gt menu 7 2 Running Programs E Instant Incubate Temperature User Name M Heated Lid GUEST H Select Blocks Select All Clear OF Cancel e Select the Temperature field and enter the desired temperature e Select Heated Lid if you are incubating at a high temperature and wish to minimize condensation refer to the Sealing with the Hot Bonnet Lid section in Chapter 4 for additional information on us ing the heated lid e Select the desired block s e Select lt OK gt The block s will now incubate at the desired temperature The Status window will display the status of the selected block Use the block selection menu to select a block Block selection menu Programs Command Tools View Utilities Block status line Incubating at 37 0 C UserName GUEST peel El Temperature Time Block 736 9 C Sample Lid 46 7 C Cycle 1 Step 00 08 17 Remaining Forever To stop the incubation select the appropriate sample block from the block selection menu Select lt Stop gt at the bottom of the window A confirmation window will appear asking you to verify termination of the instant incubation Select lt Yes gt to stop the incubation or lt No gt to
18. C for 00 03 00 Beep on Target 4 Cycle to step 2 for 29 more times 5 Incubate at 10 C forever End Note You may need to utilize the directional arrow keys to view all steps File Utilities Once you have edited a program any number of manipulations can be used to archive it for later use including saving copying deleting and moving You can also delete a folder These functions with the exception of saving can be accessed from the lt Programs gt drop down menu on the Status window menu bar Saving an Edited Program The decision required here is whether to save the program under the same or different tilename In Chapter 5 we discussed the lt Save As gt button which allows the creation of a new filename In this instance we will simply save the edited files under the same name e Select lt Save gt Your file can now be selected and reviewed for further editing Please note that in the advanced programming window utilizing the lt Save gt feature will bring you to the Status window after implementation whereas the lt Save As gt feature will bring you back to the advanced programming window Therefore if you plan on continuing to edit the file lt Save As gt would be the simpler choice 6 8 Managing and Editing Programs W Copying a Program e Select lt Programs gt in the Status window e Select lt Copy gt from the drop down menu From Folder To Folder ADVANCED BASIC ESAMPLE
19. Chapter 10 offers troubleshooting information for the DNA Engine Dyad cy cler Chapter 11 describes the installation and operation of the RAD 200 Remote Alpha Dock accessory Introduction Important Safety Information Sate operation of the DNA Engine Dyad cycler begins with a complete understanding of how the instrument works Please read this entire manual before attempting to operate the Dyad cycler Do not allow anyone who has not read this manual to operate the instrument Warning The DNA Engine Dyad cycler can generate enough heat to inflict serious burns and can deliver strong electrical shocks if not used according to the instructions in this manual Please read the safety warnings and guide lines in Appendix A and exercise all precautions outlined in them Warning Do not block the Dyad cycler s air vents see figs 2 1 and 2 4 for loca tion Obstructing air vents can lead to overheating and slightly enhanced risk of electrical shock and fire E DNA Engine Dyad Operations Manual Layout and Specifications Front View 2 2 Control Panel 2 2 Back View 2 3 Bottom View 2 3 Alpha Units 2 4 Single Block Models 2 4 Dual Block Models 2 4 Slide Block 2 4 Power Bonnet Accessory 2 4 Specifications 2 5 Gradient Specifications 2 5 M DNA Engine Dyad Operations Manual Front View Figure 2 1 Dual block Alpha unit lid closed Air intake vents Display screen Numeric keypad Touch p
20. DNA Engine Dyad thermal cycler Designed by a team of molecular biologists and engineers the DNA Engine Dyad cycler delivers multi block thermal cycling with superior thermal performance The programmable Dyad cycler with its dual bay chassis is ideal for running multiple protocols and accommodating multiple users Some of the Dyad cycler s many features include Interchangeable sample blocks the Alpha unit family accommodates a vari ety of tubes microplates and slides Hot Bonnet heated lid for oil free cycling or the Power Bonnet lid for auto mated systems Intuitive software with user friendly interface for programming editing file man agement and much more Choice of calculated temperature control for highest soeed and accuracy or of block or probe temperature control for compatibility with protocols designed for a variety of instrument types Instant Incubate feature for continuous temperature incubations Using This Manual This manual contains instructions for operating your DNA Engine Dyad cycler safely and productively Chapter 2 acquaints you with the physical characteristics of the DNA En gine Dyad cycler Chapters 3 4 present the basics of installation and operation for the DNA Engine Dyad cycler Chapters 5 6 and 7 describe the creation editing and running of pro grams Chapter 8 outlines the software utilities Chapter 9 explains the proper maintenance of the DNA Engine Dyad cycler
21. In addition some protocols can include a sustained incubation at sub ambient temperatures to preserve the integrity of the products We will choose an initial incubation at 94 C for 1 minute before cycling and a final incuba tion of the sample at 10 C forever Your written program might now appear as follows Raw program Use calculated temperature control mode Use constant lid control mode at 100 C An initial incubation at 94 C for 1 minute ii 92 C for 30 seconds 2 Gradient from 45 C to 65 C for 3 minutes 3 Goto step 1 29 more times 4 An incubation at 10 C forever 9 END Now that we ve made some important decisions regarding the implementation of our pro gram we are ready to begin entering steps 5 13 M DNA Engine Dyad Operations Manual 5 14 Entering Program Steps From our example we are ready to enter a new program When executing a selected command via the touch pad we will use the method of tapping the pad with a fingertip This can also be accomplished with the left button below the touch pad as well as the lt lt EN TER gt gt button on the numeric keypad If the external mouse device has been enabled this corresponds to left clicking the mouse Start up procedures for the Dyad cycler are covered in detail in Chapter 4 including start up screens Please review Chapter 4 before proceeding with the entering of program steps The Status Window Once the Dyad cycler has completed its boot up seq
22. Lid Constant window will appear 5 16 Creating Programs W Lid Constant E4 Maintain lid temperature at c Turn off lid when block temperature drops OF Cancel Place the cursor in the Maintain lid temperature at field and select the field We have decided to set the lid to a constant temperature of 100 C Enter 100 from the numeric keypad Place the cursor in the below field and select the field We have decided to turn the lid off when the block drops below 30 C Enter 30 from the numeric keypad Select lt OK gt We have returned to the Mode Selection window Select lt OK gt We have returned to the graphical programming window It is from this location that you will add steps using the lt Temp gt lt Gradient gt and lt Goto gt options Additionally buttons running across the window bottom provide options to lt De lete Step gt lt Save Run gt lt Save gt or lt Save As gt programs and lt Cancel gt the current programming session Using the File Save As Window The lt Save gt and lt Save As gt buttons are probably the most important buttons in the graphical programming window since a program that is saved can be used or edited at a later date Please note that a new graphical program must be renamed using the lt Save As gt feature prior to initiating a run TEMPLATE is not a valid name nor can a graphi cal program with the default name TEMPLATE be run This restriction is de
23. Once you have created a number of programs you may want to create separate folders to organize them Perhaps you will use separate folders for different us ers or experimental series To copy a program e Highlight the From Folder and To Folder locations for the copy e Highlight the program in the Copy Program list to be copied e Select lt Copy gt The program now resides in the destination folder e Select lt Exit gt to return to the Status window E DNA Engine Dyad Operations Manual Deleting a Program e Select lt Programs gt in the Status window e Select lt Delete gt from the drop down menu Folder Program Listing of ADYVHI Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 0 C for 00 01 00 Beep on Target 2 Incubate at 92 0 C for 00 00 30 Beep on Target LA Er A Hil 45 Important Use caution when deleting programs as deletions are irreversible and the delete program window is very similar to the open program window We provide a conve nient program listing at the bottom of the window so that you can determine whether you truly wish to delete the program e Highlight the Folder containing the program to be deleted e Highlight the Program e Select lt Delete gt Before a program is deleted you are presented with a confirmation screen asking you to verity the deletion Are You Sure You Want to Delete ADYHT 7 If you wish to delete t
24. System Performance 10 9 Problems with an Alpha Unit 10 10 Problems Related to Protocols 10 10 Problems due to Environmental Conditions Setup and Maintenance 10 13 M DNA Engine Dyad Operations Manual 10 2 Sources of Problems The Dyad cycler is designed to handle even the most stringent thermal cycling requirements A major strength of the Dyad cycler is its flexibility which allows multifaceted and demand ing cycling protocols to be implemented with the greatest of experimental and programming ease However occasional problems may still be encountered Problems can result in a number of ways We group them as follows e System problems e Problems in power up e Problems in system performance e Problems with Alpha units e Problems due to environmental conditions setup and maintenance e Problems related to protocols When troubleshooting a difficulty it is advisable to have the following pieces of information available should it become necessary to contact MJ Research Inc for assistance e Serial and catalog numbers for the base as well as Alpha units e Exact nature of the problem e Frequency of the problem i e is the problem repeatable e Steps already taken to troubleshoot the problem i e controls performed System Problems The Status window was reviewed in Chapters 5 6 and 7 covering the creation editing and running of programs For a review of the basic menus of this window please review these c
25. Utilities 6 8 Saving an Edited Program 6 8 Copying a Program 6 9 Deleting a Program 6 10 Moving a Program 6 11 Deleting a Folder 6 11 E DNA Engine Dyad Operations Manual In the previous chapter various entering and editing features were discussed as they applied to entering a graphical and or an advanced program In this chapter we cover in more depth the options available for the manipulation of existing Dyad programs The programming conventions listed in Chapter 5 will also be used here Please review these before proceeding Opening a Program Once a program has been saved to disk as the programs GRAPH 1 and ADV 1 were in the previous chapter it can then be opened for editing or running From the Status window menu bar e Select lt Programs gt e Select lt Open gt An additional menu appears allowing you to choose lt Advanced Mode gt or lt Ba sic Mode gt Opening a Program in Advanced Mode To open our example advanced program ADV 1 e Select lt Advanced Mode gt Folder Listing of ADYHI Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 0 C for 00 01 00 Beep on Target 2 Incubate at 92 07C for 00 00 30 Beep on Target adier om 45 You are presented with the open program window In this window the Folder field lists the available folders and the Program field lists all programs available in the currently selected folder A Listing of the
26. Y Y VY SU et ESENES EACEA EAKA EAESES ESEAS fete ff KAKARA RARES ESEAS ff ff Es HE mE HE MEZ 4 14 Operation W Appendix 4 B Safety Warning Regarding Use Of 35S Nucleotides Some researchers have experienced a problem with radioactive contamination when using S in thermal cyclers This problem has occurred with all types of reaction vessels The Problem When S nucleotides are thermally cycled a volatile chemical breakdown product forms probably SO This product can escape the vessel and contaminate the sample block of a thermal cycler and possibly the air in the laboratory Contamination has been reported with microassay plates 0 2ml tubes and 0 5ml tubes 96 Well Polycarbonate Microplates These microplates present the largest risk of contamination Polycarbonate is somewhat permeable both to water and the 35S breakdown product This problem is exacerbated when polycarbonate plates are held at high temperatures for long periods of time or when the plates are sealed for oil free thermal cycling 0 2ml Polypropylene Tubes and Polypropylene Microplates These tubes are manufactured with very thin walls to enhance thermal transfer The thin walls are somewhat fragile and can craze or develop small cracks when subject to mechanical stress Undamaged thin polypropylene tubes may also be somewhat perme able to the 35S breakdown product Either way there have been reports of S passing thr
27. Yes gt to open the program in advanced mode 6 3 E DNA Engine Dyad Operations Manual 6 4 Opening a Program in Basic Mode You can choose to open and edit a graphical program in either the graphical programming window or in the advanced programming window Opening a graphical program in ad vanced mode is desirable if you wish to add step modification options incubations below O C or other programming features not available in graphical programs see the Types of Programs section in Chapter 5 for a listing of available program features However once advanced only features are added to a graphical program the program can no longer be opened or edited in basic mode We will open our example graphical program GRAPH 1 in basic mode The procedure is similar to that described above for opening a program in advanced mode and is summa rized here e Select lt Programs gt e Select lt Open gt e Select lt Basic Mode gt The open program window will appear e Select the folder FOLDER e Select the program GRAPH 1 e Select lt OK gt GRAPH 1 will be displayed in the graphical programming window Editing a Program Recall our general program Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target advanced only 2 Incubate at 92 C for 00 00 30 Beep on Target advanced only 3 Gradient from 45 C to 65 C for 00 03 00 4 Cycle to step 2 for 29
28. figure 2 2 includes a display screen cursor keys a numeric keypad with enter key and a touch pad with left and right mouse buttons Display Screen e The display screen is a 1 4 size VGA screen for displaying thermal cycler conditions and programs Display screen contrast adjustment Underneath the front lip of the Dyad cycler positioned below the cursor buttons there is a small partially recessed knob see figure 2 4 This knob can be rotated to optimize the contrast of the color display Operation Keys e Cursor keys left right up and down arrows Use to move around within the display screen e Numeric keypad Use to enter numeric values e Enter key below keypad Use to accept specific programming additions and modifications e Touch pad Use to move the display screen pointer with the movement of a finger tip Once the pointer is positioned over a menu or selection item on the display screen a single or double tap of the touch pad with a fingertip will implement the command A tap on the touch pad corresponds to clicking the left button on a mouse e Mouse buttons left and right buttons below touch pad Pressing the left button is identical to a single tap on the touch pad or clicking the left button of a mouse The right button has no current function 4 3 m DNA Engine Dyad Operations Manual 4 4 Block Status Lights e When illuminated these blue lights indicate whether the left and or r
29. for unusual circumstances that may require it Ordinarily though it should be used with caution While the Dyad cycler will have no trouble heating the probe to the target temperature if the probe is seated or prepared differently from the sample tubes actual sample temperatures can vary widely from the probe s temperature Probe control cannot be used with microplates or slides or in conjunction with the heated lid Modifying Block and Probe Control Programs for Calculated Control Probe control programs will generally run well under calculated control with no modifi cation other than changing the method of temperature control Block control programs can be changed to calculated control by subtracting at least 15 20 seconds from each temperature step Some empirical testing may be required to adjust modified programs for optimum performance We generally recommend not reducing the incubation time for a step below 5 seconds while in calculated control mode Creating Programs W Modifying a Program Designed for a Different Machine The ramp programming step can be used to adapt programs designed for thermal cyclers with slower maximum heating and cooling rates than the Dyad cycler In addition a given protocol will occasionally work better with a slower rate of temperature change the ramp step can be used to optimize the program for such a protocol Choosing a Lid Control Mode When a sample is heated condensation on the tube cap
30. in scenario 3 and in the Editing Step Parameters section to alter the parameters of the displayed step If step two is not a temperature step e Select step 1 e Select lt Temp gt A new temperature step will be added after step 1 with the default temperature and time parameters Follow the instructions in scenario 3 and in the Editing Step Parameters section above for information on altering the step parameters 5 23 M DNA Engine Dyad Operations Manual 5 24 Step two of our protocol should now specify a temperature incubation of 92 0 for a duration of 00 30 Step 2 Program GR4PH i E Lid Mode CONSTANT 100 0C temperature step ma 00 30 O0 30 OO 30 Add Step after Control Mode cac Grad Golo Forever Entering a Gradient Step Step three of our program is a gradient step designed to determine the optimal anneal ing temperature of our oligonucleotide If step three of the displayed protocol is not a gradient step two black lines e Select step two e Select lt Grad gt A new gradient step should now appear as step three with the default higher tempera ture limit of 65 C and the default lower temperature limit of 50 C The default duration is 30 seconds The maximum temperature range for a gradient is 24 C and the minimum is 1 C Frac tional degrees are not accepted Recall that in our example protocol the gradient step should specify a range of 45 C to 65
31. is a check box for enabling syntax checking This will filter out any commands that do not conform to the PTC Remote Command Set syntax Attach a null modem serial interface cable MR 02371 or equivalent to the RS 232 port connector on the back of the Dyad cycler Connect to either COMM1 or COMM2 on a PC or the serial port on a Mac The Dyad cycler is now ready to be run in remote mode Important Do not exit remote mode while a protocol is running If a protocol is loaded and run remotely ending the remote session by exiting the Remote Mode window will cancel this protocol A confirmation screen will ask you to confirm your intent to exit the Remote Mode window Log on to www mijr com for the latest syntax of the remote command set lt Auto Remote gt Select lt Auto Remote gt from the lt Utilities gt menu to direct the Dyad cycler to automatically enter remote mode upon subsequent power up This feature is particularly useful if you consistently control the Dyad cycler using a desktop computer It eliminates the need to select lt Remote gt from the lt Utilities gt menu after every power up To disable the auto remote feature first select lt Exit gt to exit remote mode see the warning in the lt Remote gt section above The cycler will return to the standard operation mode and the Status window will be displayed Select lt Auto Remote gt from the lt Utilities gt menu such that a check mark no longer appears in front of the
32. operation with small volumes gt 5ul Alpha Units and the Remote Alpha Dock System E The 30 48 Dual Block for 0 5ml amp 0 2ml Tubes ALD 1234 For those who wish to have available both the O 5ml format and the 0 2ml format simulta neously a combination Alpha unit is offered This dual unit has two independent blocks with two different formats one holds 30 x 0 5ml tubes and the other 48 x O 2ml tubes or a 48 well plate Each block has an integral heated lid This Alpha unit has quickly become one of the most popular MJ Research units The 16 16 Twin Towers Alpha unit for Glass Slides ALD 0211 DNA En gine Dyad cyclers ALD 0212 Tetrad cycler This dual unit has two independent blocks each of which can hold 16 slides in isothermal chambers that ramp at rates up to 1 2 C second Slides can easily be sealed with either Self Seal reagent or Frame Seal chambers and the blocks can double as humidified chambers for hybridizations The 96 with a Power Bonnet motorized lid ALP 1296 This sample format can hold either one 96 well microplate or 96 x O 2ml tubes or strips of O 2ml tubes and features a Power Bonnet motorized lid for remote control of lid opening and closing The 384 with a Power Bonnet motorized lid ALP 1238 This sample format can hold one 384 well microplate and features a Power Bonnet motor ized lid for remote control of lid opening and closing Contact MJ Research Inc at 888
33. or plate cover can take place This changes the volume of the sample the concentration of components and the kinetics of the enzymatic reaction The Hot Bonnet heated lid minimizes condensation by heating the upper surface of the reaction vessel to a temperature slightly greater than that of the sample itself The Dyad cycler can control lid temperature in three possible ways Constant Track ing or Off Constant Mode This mode maintains the inner lid surface at a specific temperature regardless of sample temperature When using constant mode specify a lid temperature at least 5 C higher than any temperature used in the protocol Note We will choose to maintain a constant lid temperature of 100 C in our example program Tracking Mode Offsets the temperature of the heated inner lid a minimum specified number of degrees Celsius in comparison to the temperature of the sample block Track ing is useful for protocols with long incubations in the range of 30 70 C where it may be undesirable to keep the lid at a very high temperature An offset of 5 C above block temperature is adequate for most protocols Off No power is applied to the heated lid In this mode condensation will occur at a rate consistent with the incubation temperature and the type of tube or plate sealant being used This option is recommended only when using an oil or wax overlay Choosing a Temperature Ramping Rate Advanced Mode Only Fast thermal ramping bet
34. steps in the currently highlighted program appears near the bottom of the window Managing and Editing Programs W We had previously saved ADV 1 in the FOLDER2 folder Select the folder FOLDER2 Select the program ADV 1 Select lt OK gt Program ADYHI Temperature Control Mode Calculated 1o Lid Control Mode Constant at 100 C 1 Incubate at 94 0 C for 00 01 00 Beep on Target 2 Incubate at 92 0 C for 00 00 30 Beep on Target 3 Gradient from 45 C to 65 C for 00 03 00 4 Cycle to step 2 for 29 more times 5 Incubate at 10 0 C forever alt Add Step After Temp Gradient Golo Lid Delete Step Save Run Save Save As Cancel You are presented again with the advanced programming window It is from this window that steps can be inserted deleted or edited While all graphical programs can be opened and edited in advanced mode only a subset of advanced programs can be opened and edited in basic mode Advanced programs that meet the criteria outlined for graphical programs in the Types of Programs section of Chapter 5 can be opened in basic mode Our advanced program ADV 1 can not be opened in basic mode because it con tains the step modification option Beep on Target If an advanced program can not be opened in basic mode the following message will appear This file has options not available to the Basic Hode Editor Do you want to open the File with the Advanced Mode Editor Select lt
35. the Higher Temperature of the gradient range as well as the gradient hold Time The maximum gradient range is 24 C and the minimum range is IC Below the Extend Time option allows for an increase or decrease in incubation time per cycle similar to the option available for temperature steps Since our hypothetical target is 60 C we will choose 45 C for the lower limit and 65 C for the higher limit Additionally we will enter 3 minutes for the incubation hold time Select the Lower Temperature field Select the Higher Temperature field Select the Time Min field 5 33 M DNA Engine Dyad Operations Manual 5 34 The newly created gradient can be previewed graphically Select lt Preview gt Gradient Preview This distribution of temperatures specified by the gradient should be reviewed and any changes to the gradient limits made before a program run Please note that the gradient temperature differential is not linear with a broader spread in temperature between the center columns of wells This is a consequence of the geometry of the Peltier Joule heaters that underlie the block and is normal Rest assured that the temperatures displayed are quite accurate for each well in that column 0 4 C of actual column temperature Select lt Close gt Select lt OK gt The program listing should now appear as follows Since the program step listing field is limited please use the directional arrow keys to navigate
36. the program step as included in a protocol to appear as fol lows Incubate at 60 0 C for 00 00 30 Decrease by 0 2 C every cycle However such a step is not needed in our protocol so we will continue onto the next step the sustained incubation We will include an incubation at 10 C forever to preserve our sample integrity The selections are similar to adding a temperature step with the exception of selecting Forever rather than entering an incubation Time The final program should appear as follows Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target 2 Incubate at 92 C for 00 00 30 Beep on Target 3 Gradient from 45 C to 65 C for 00 03 00 4 Cycle to step 2 for 29 more times 5 Incubate at 10 C forever End The program is now finished Select lt Save gt to ensure that your work is pre served In Chapter 6 we will learn to edit the various programming steps to include different parameters and in Chapter 7 we will learn how to actually run our program M DNA Engine Dyad Operations Manual 5 40 6 Managing and Editing Programs Opening a Program 6 2 Opening a Program in Advanced Mode 6 2 Opening a Program in Basic Mode 6 4 Editing a Program 6 4 Editing a Graphical Program 6 5 Editing an Advanced Program 6 6 Highlighting and Selecting Program Steps 6 6 Deleting a Step 6 6 Inserting a Step 6 7 Editing a Step 6 7 File
37. through the steps to review them Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target 2 Incubate at 92 C for 00 00 30 Beep on Target 3 Gradient from 45 C to 65 C for 00 03 00 End Creating Programs W As discussed previously increasing the time per cycle of a temperature step might be desirable in some protocols where extra time is required during later cycles to allow synthesis to be completed We will not require such a step in our cycle sequencing protocol but describe it here for completeness The Extend Time Option This programming option progressively extends an incubation step with each subsequent cycle This is typically used during an extension step to allow for diminishing activity of an enzyme or to allow an enzyme to do its job among an ever increasing quantity of product Assume for example that we required a 60 C step with a 1 minute incubation and that we wished to increase the incubation time by 5 seconds per cycle In the Temperature Step window you would implement the following e Enter 60 C in the Temperature field e Enter 1 in the Time Min field e Select Extend Time e Select lt Set Parameters gt for the Extend Time option The Extend Time window appears Extend Time E M Increase Decrease by I seconds per cycle OF Cancel e Select Increase e Position the cursor over the by __ seconds per cy
38. through the crys talline blocks and return to a low energy state at the other conductor semiconductor interface with the re lease of the previously absorbed heat A thermal gra dient of up to 70 C can be generated across the blocks in this manner AT o ey N type bismuth telluride amp Ptype bismuth y telluride Power input The direction of heat pumping is reversed by reversing the polarity of current flow through the thermoelectric module and the amount of heat pumped is changed by changing the amount of current passed Both direction and amount of current flow are dictated by a microprocessor allowing precise control of thermal cycling in the Alpha unit block Appendix C Shipping Instructions W Appendix C Shipping Instructions for US Residents Users residing in the United States should follow these instructions for shipping a machine to MJ Research for factory repair or an upgrade Users outside of the United States should send machines to their distributor in accordance with shipping instructions obtained from the distributor 1 Call MJ Research 888 652 9253 to obtain a return materials authorization RMA num ber Machines returned without an RMA will be refused by the Receiving Department 2 Thoroughly clean the machine removing excess oil and radioactive and other biohazardous substances To protect the health of our employees MJ Research will not repair or upgrade any ma
39. to rest your finger on the touch pad as you may unintentionally select a field on the display Note If you have chosen to enable the external mouse device see the Setting Up the DNA Engine Dyad Cycler section in Chapter 3 the touch pad can not be used to input program ming commands The cycler will recognize either the touch pad OR a mouse but not both input devices simultaneously Touch Pad Buttons These two buttons are located below the touch pad Once the pointer is positioned via the touch pad over a menu or selection item on the display screen the left button can be used to implement commands Pressing this button has the same effect as a single tap on the touch pad Note The right touch pad button is reserved for future programming functions 5 3 M DNA Engine Dyad Operations Manual 5 4 Programming Conventions Before starting the Dyad programming process let s review some common ground conven tions used here e lt lt gt gt will be used to indicate actual keys on the control panel such as lt lt ENTER gt gt lt lt gt gt and lt lt LEFT gt gt e lt gt will be used to indicate windowed menu items or buttons such as lt PROGRAMS gt lt RUN gt and lt TOOLS gt e Italics will be used to indicate windowed items that are not drop down menu items or buttons such as Calculated Block and Tracking Typically these will be parameter selection items e Select is meant to be syno
40. 1 Engine 2 3 11 ROMD 6 22 WOS Build 932 This screen indicates the Dyad system software version The software version information on this screen can be used to determine if an update is necessary Select lt OK gt to exit from this screen 8 2 Using the Utilities E lt User Name gt When this item is selected the following screen will appear User Name GUEST GUEST Add User Name i OK Cancel By selecting lt Add User Name the virtual keyboard will appear allowing users to enter their name prior to program execution In this manner any potential user can determine the owner of a block the person running a program as their name will be displayed when the block is selected in the Status window To delete a user name highlight the name and select lt Delete User Name gt Select lt OK gt to exit from this screen lt Set Date Time gt Choosing this option will allow the setting of the Dyad system time Set Date Time Month Day Year Hrs Min Sec 23 59 59 Cancel Time Enter the appropriate information in the fields provided and select lt OK gt M DNA Engine Dyad Operations Manual 8 4 lt Remote gt To operate a Dyad cycler in remote mode i e from a desktop computer select lt Remote gt from the lt Utilities gt menu The Remote Mode window will appear on the screen Remote Mode Syntax Checking Clear The only option available
41. 4 0 C e Enter 94 from the numeric keypad e Tap or click once to accept the change e Position the cursor text pointer in the time minute field of step 1 and select by tapping clicking once e Use the backspace key to delete the current value if it is not 01 e Enter O1 from the numeric keypad and tap or click once to accept the change e Position the cursor text pointer in the time second field of step 1 and select by tapping clicking once e Use the backspace key to delete the current value if it is not 00 e Enter 00 from the numeric keypad and tap or click once to accept the change Creating Programs W Step one of our protocol now consists of a temperature step with an incubation tempera ture of 94 0 and a duration of 01 00 Program GRAFHAt E Lid Mode CONSTANT 100 0 Step temperature step Cr 55 0 OO 30 00 30 OO 30 Add Step after Bi Control Mode cac Temp Grad Golo l Forever Delete Step Save Save As Save Hun Cancel Recall that the maximum programmable temperature is 105 0 C and the minimum pro grammable temperature is 0 0 C in a graphical program The maximum duration of a temperature step in a graphical program is 99 minutes and 59 seconds or forever Step two of our program is also a temperature step but with an incubation temperature of 92 0 C and a duration of 30 seconds If step two in the displayed protocol is a temperature step follow the instructions
42. Appendix D VWGr Ones sesucine eon a Nr er D 1 UN Se pec E E I EE EE A T E re In 1 Declaration of COMO IY 5si24 est ananas DoC Documentation Conventions Before describing the various features of the Dyad cycler let s define some common ground conventions lt lt gt gt will be used to indicate actual keys on the control panel such as lt lt ENTER gt gt lt lt gt gt and lt lt LEFT gt gt lt gt will be used to indicate windowed menu items or buttons such as lt PROGRAMS gt lt RUN gt and lt TOOLS gt Italics will be used to indicate windowed items that are not drop down menu items or buttons such as Calculated Block and Tracking Typically these will be parameter selection items Select is meant to be synonymous with click on point and click and any phraseol ogy implying selection of menu or option items with a mouse Particularly with the Dyad cycler select should symbolize any physical selection on the Dyad input devices touch pad mouse numeric keypad arrow keys to access one of the user interface windows This includes single or double taps on the touch pad pressing of the left touch pad button or pressing the lt lt Enter gt gt button on the numeric keypad Introduction Meet the DNA Engine Dyad Cycler 1 2 Using This Manual 1 2 Important Safety Information 1 3 E DNA Engine Dyad Operations Manual Meet the DNA Engine Dyad Cycler Thank you for purchasing an MJ Research
43. C and a duration of 3 minutes e Position the cursor text pointer in the higher limit temperature field and select by tapping clicking once e Use the backspace key on the numeric keypad to delete the higher temperature if it is not 65 C e Enter 65 from the numeric keypad e Repeat the steps above for the lower temperature limit entering a value of 45 from the keypad Creating Programs W e Tap or click once to accept the changes to the gradient range Note Change both the higher and lower temperatures before accepting the changes to the gradient step to ensure that the temperature differential is not greater than 24 C or less than 1 C e Position the cursor text pointer in the time minute field of step 3 and select by tapping clicking once e Use the backspace key to delete the current value if it is not 03 e Enter 03 from the numeric keypad and tap or click once to accept the change e Position the cursor text pointer in the time second field of step 3 and select by tapping clicking once e Use the backspace key to delete the current value if it is not 00 e Enter 00 from the numeric keypad and tap or click once to accept the change Entering a Goto Step Step 4 of our program incorporates a goto step designed to cycle a portion of the program a predetermined number of times We have chosen to cycle back to step 2 and repeat steps 2 and 3 an additional 29 times A graphical program can only contain one go
44. Canada or your local distributor 7 ts may have been outside the US or Canada Please observe the following guidelines 1 place instruments at least 10cm apart 2 avoid lacing instruments in any ined where hot air might enter the intake vents 3 place Th o ture is instrument on a hard surface with aeons ee ig a Ino debris or paper underneath somewhat higher than norma 4 clean all air vents and Alpha PSO CAF The power supply sensor will be i fins of dust and d bris es and you will be sal An Fe me ne notified if any problem is found 221 running He instrument in y P areas with ambient temperatures above 25 C Continue to use the instrument If problem reoccurs please call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada 10 5 M DNA Engine Dyad Operations Manual Error Code Please observe the following guidelines 1 place instruments at least 10cm apart 2 avoid oh instruments in any The power supply temperature ocation where hot air might ji eee sf T enter the intake vents 3 place a T S R ead instrument on a hard surface with prevent instrument damage the debris or paper underneath PSO PT Sa O Rare A A clean all air vents and Alpha stopped Overheating od Unit fins of dust and debris 5 cas by ee airtlow or avoid running the instrument in i Sm win Me Powel areas with ambient temperatures iad ca above 25 C Do not use the instrument until y
45. Control 5 10 In Sample Probe Control 5 10 Modifying Block and Probe Control Programs for Calculated Control 5 10 Modifying a Program Designed for a Different Machine 5 11 Choosing a Lid Control Mode 5 11 Choosing a Temperature Ramping Rate Advanced Mode Only 5 11 Choosing a Temperature Hold Time 5 12 Choosing A Thermal Gradient 5 12 Beyond the Example Protocol Other Considerations 5 13 Creating Programs Entering Program Steps 5 14 The Status Window 5 14 Entering a Program Using Graphical Mode 5 15 Using the Mode Selection Window 5 16 Using the File Save As Window 5 17 The Graphical Programming Window 5 19 Selecting a Step 5 20 Editing Step Parameters 5 20 Deleting a Step 5 20 Adding a Step 5 20 Entering a Temperature Step 5 21 Entering a Gradient Step 5 24 Entering a Goto Step 5 25 Entering a Forever Incubation 5 26 Entering a Program Using Advanced Mode 5 28 Entering a Temperature Step 5 30 Entering a Gradient Step 5 33 The Extend Time Option 5 35 Entering a Goto Step 5 36 Entering a Lid Control Step 5 37 The Slow Ramp Option 5 37 The Increment Temp option 5 38 M DNA Engine Dyad Operations Manual In this chapter we will revisit the setup of the front panel specifically those items used in program input We will describe the conventions used as well as the various programming steps and what they accomplish We make suggestions regarding the translation of a cycle sequen
46. In Did you hear a beep y Are Alpha units in Secure Alpha 1 the correct position units and are handles locked in place Does the initialization screen appear Are fuses Replace fuses lt good 5x20mmMm 6 3A Unit defective return for service Was 1 hit to enter Selftest Selftest Does the About screen appear ry Unit ready 10 8 Troubleshooting I Problems with System Performance Should you encounter problems with menu navigation front panel manipulation or any performance aspect of a Dyad cycler that has successfully powered up please follow the steps in this flowchart to determine the best course of action Again a problem should be a consistent repeatable problem Figure 10 2 System Performance Troubleshooting Flowchart Unit ready Do a een nue aes des Select 1st block Re boot and run self test program Are temperatures indicated on the display Front end software may have become corrupted Re load software Re Test Select last block Are temperatures indicated on the display Problem corrected Unit defective return for service Are the number keys working Try an Instant Incubate Are the cursor keys working Try navigating a window with them Is the touch pad working
47. PTC 220 DNA Engine Dyad Peltier Thermal Cycler Operations Manual Version 1 3 1 AXA MJ Research INCORPORATED Boston San Francisco Tahoe Copenhagen Seoul Copyright 2002 MJ Research Incorporated All rights reserved Reproduction in any form either print or electronic is prohibited without written permission of MJ Research Incorporated 05570 revA A DNA Engine DNA Engine Tetrad DNA Engine Dyad Dyad Alpha Hot Bonnet Power Bonnet Multiplate Chill out Self Seal Remote Alpha Dock MJ Research marks and helix logo are trade and or service marks belonging to MJ Research Incorporated Microseal Hard Shell and Twin Towers are registered trademarks belonging to MJ Research Incorporated Datalight is a registered trademark of Datalight Inc ROM DOS is a trademark of Datalight Inc WinLight is a trademark of Datalight Inc Copyright 1989 2000 Datalight Inc All Rights Reserved Thermal cyclers can be used for a number of purposes including the polymerase chain reaction PCR PCR is covered by patents owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd who have granted exclusive and nonexclusive licenses for some types of applications Roche and its licensees provide end user licenses within their respective fields These licenses have different terms depending on the particular application of PCR and different rules may apply in different countries Anyone who intends to use MJ Research equi
48. ad Control Panel Figure 2 2 OO O Q D O LOLO MJ Research 9 GYOO o rm 1 4 VGA display screen Cursor keys Keypad Touch pad Touch pad mouse keys 2 2 Layout and Specifications W Alpha unit icw Back V Figure 2 3 A LER eS S L Power switch RS 232 port Ethernet port Fuses Power cord jack 8 amp gt Ea F 0 D ao External mouse port External mouse touch pad Screen contrast adjustment knob selection switch Air intake vents SIDA F Dyad cycler rear 2 3 M DNA Engine Dyad Operations Manual Alpha Units Single Block Models 60 Single Holds 60 x 0 5ml tubes 96 Single Holds 96 x 0 2ml tubes or one 96 well microplate 384 Single Holds one 384 well microplate Flat Block Holds customer designed adapter through four screw down points Dual Block Models 30 30 Dual Holds 2 x 30 x 0 5ml tubes 30 48 Dual Holds 1 x 30 x 0 5ml tubes and 1 x 48 x 0 2ml tubes 48 48 Dual Holds 2 x 48 x O 2ml tubes or half plates Slide Block ser 1 slides Twin Towers Holds 2 x 16 standard Power Bonnet Accessory Permits remote control of Alpha unit lid opening available for Alpha unit models 96 384 and flat block 2 4 Specifications Thermal r
49. and closed repeatedly without readjustment as long as neither the tube or microplate type nor the sealing method is changed Any change in vessel type or sealing method requires readjustment of the Hot Bonnet lid Follow these steps to adjust the pressure exerted by the inner lid 1 Make sure the block s wells are clean Even tiny amounts of extraneous material can decrease thermal conduction and interfere with the proper seating of a microplate or tubes 2 Open the Hot Bonnet lid Turn the blue thumbwheel all the way counterclockwise to completely raise the inner lid 3 Load either a microplate or at least eight individual tubes into the sample block The inner lid pivots around a central point so it is important to distribute individual tubes evenly load at least four tubes in the center of the block and at least one tube in each of the four corners of the block If using a sealing film or mat apply it to the loaded microplate according to the manufacturer s directions 4 Close the Hot Bonnet lid by pressing down on the top lever Turn the thumbwheel clockwise to lower the inner lid onto the loaded microplate tubes The thumbwheel turns easily at first since the inner lid has not yet come into contact with anything Stop turning the thumbwheel when you feel increased resistance which indicates that the inner lid has touched the microplate tubes 5 For microplate sealing films or mats that require additional pressure turn the thum
50. ange Accuracy Thermal uniformity Ramping speed Sample capacity Line voltage Frequency Power Fuses Displays Ports Memory Weight Size Layout and Specifications W 5 0 to 105 C but no more than 30 C below ambient temperature 4 C to 105 C but not more than 23 C below ambient temperature for the Twin Towers unit 0 3 C of programmed target 90 C NIST traceable 0 4 C well to well within 30 seconds of arrival at 90 C for most Alpha units see specifications for individual Alpha units Up to 3 C sec for all single and dual block Alpha units Up to 1 2 C sec for the Twin Towers unit Varies with installed Alpha unit 200 240VAC 50 60Hz 1600W maximum Two 6 3A 250V 5 x 20mm One 1 4 size VGA screen 320x240 16 colors One 9 pin RS 232 serial port One ethernet port 8 MB 11kg base only 48 x 29 x 15cm I x w x h base only Gradient Specifications 96 Alpha unit only Accuracy Column uniformity Calculator accuracy Lowest programmable temperature Highest programmable temperature Temperature differential range for gradient 0 4 C of programmed target at end columns 30 seconds after the timer starts for the gradient step NIST traceable 0 4 C well to well within column within 30 seconds of reaching target temperature 0 4 C of actual well temperature 30 C 105 C 1 24 C 2 5 M DNA Engine Dyad Operations Manual 2
51. aphical 5 6 Programming conventions 5 4 Programs menu 6 8 Protocols adjusting for sample vessel type 4 6 Radioactive contamination choosing microplates and tubes 4 15 cleaning 4 16 problem with 35S nucleotides 4 15 solutions to 5S problem 4 15 Ramping rate 2 5 5 11 Receive files 4 2 8 8 Remote Alpha Dock system air supply requirements 11 7 environmental requirements 11 5 installation 11 7 operation 11 8 packing checklist 11 5 power supply requirements 1 1 6 Remote operation 8 4 Remote utility 8 4 Removing microplates See Microplates Resuming a run 7 8 Robotics installation requirements 3 5 RS 232 port location of 2 3 Run initiating 4 7 6 pausing resuming 7 7 7 8 skipping a step 7 8 status 6 7 7 terminating 7 7 Run program window 7 5 Running multiple programs 7 8 Running programs 7 4 7 6 In 8 S Safety general instructions 1 3 guideline for safe use A 2 Use of 35S Nucleotides 4 15 warnings A Sample vessels ensuring good thermal contact 4 10 loading into block 4 10 sealing reason for 4 8 selection chart 4 14 with Hot Bonnet lid and caps film 4 8 with oil or wax 4 8 selection 4 6 0 2ml tubes 4 7 O 5ml tubes 4 7 microplates 4 7 selection chart 4 14 thin vs thick walled 4 7 Use of oil to improve thermal contact with block 4 10 Savetrun 4 Saving a program 5 17 5 19 Saving edited programs 6 8 Selftest 4 2 Send files 4 2 8 8 Service utility 8 8 Setting machine up 3 2 See also Alpha units insta
52. atus Lights 4 4 Using the Data Ports 4 4 Operating Alpha Units 4 4 Installing an Alpha Unit 4 4 Removing an Alpha Unit 4 5 Opening an Alpha Unit 4 6 Closing an Alpha Unit 4 6 Selecting the Correct Sample Vessel 4 6 O 5ml Tubes 4 7 Thin Walled vs Thick Walled Tubes 4 7 O 2ml Tubes 4 7 Microplates 4 7 Sealing Sample Vessels 4 8 Sealing with Oil or Wax 4 8 Sealing with the Hot Bonnet Lid 4 8 Adjusting the Hot Bonnet Lid s Pressure 4 9 Loading Sample Vessels into the Block 4 10 Using Oil to Thermally Couple Sample Vessels to the Block 4 10 Using the Optional Probe 4 11 Customizing the Probe Vessel 4 11 Adding the Oil 4 12 Loading and Connecting the Probe 4 12 Detecting a Faulty Probe 4 13 Appendix 4 A Tube Microplate and Sealing System Selection Chart 4 14 Appendix 4 B Safety Warning Regarding Use Of 35S Nucleotides 4 15 Appendix 4 C Using Silicone Oil in the Probe Tube 4 17 m DNA Engine Dyad Operations Manual 4 2 Turning the DNA Engine Dyad Cycler On Caution Do not insert or remove an Alpha unit with the DNA Engine Dyad cycler turned on electrical arcing can result Read the safety warnings in Ap pendix A regarding electrical safety before inserting or removing an Al pha unit or operating the Dyad cycler The Alpha units must be installed prior to Dyad cycler power up see the Operating Alpha Units section below for installation instructions The power switch is located at
53. bwheel clockwise an extra half turn past the point of initial contact to set an appropriate lid pressure Caution Do not turn the thumbwheel more than three quarters of a turn This can make it hard or impossible to close the lid and puts excessive strain on the latch holding the lid closed A 9 m DNA Engine Dyad Operations Manual An extra half to three quarters of a turn ensures the correct pressure for most types of reaction vessels Some empirical testing may be required to determine the optimum pressure required for certain vessels Once this pressure has been determined the thumbwheel position may be marked with a colored marking pen or piece of tape Note As an aid in gauging how much the thumbwheel has been turned mark it at the quarter turn positions or every sixth bump on the thumbwheel there are 24 total bumps Loading Sample Vessels into the Block When using a small number of tubes load at least one empty tube in each corner of the block to ensure that the Hot Bonnet lid exerts even pressure on the sample tubes see Adjust ing the Hot Bonnet Lid s Pressure above To ensure uniform heating and cooling of samples sample vessels must be in complete contact with the block Adequate contact is ensured by always doing the following e Ensure that the block is clean before loading samples see Chapter 9 for cleaning instruc tions e Firmly press individual tubes or the microplate into the block
54. cause there are no temperature com mands or incubation times to constitute an actual run Recall again our raw example program Use calculated temperature control mode Use constant lid control mode at 100 C An initial incubation at 94 C for 1 minute l 92 C for 30 seconds 2 Gradient from 45 C to 65 C for 3 minutes 3 Goto step 1 29 more times 4 An incubation at 10 C forever 5 END The first actual step in the protocol is the incubation at 94 C for 1 minute We will use the maximum rate of temperature ramping to this step and we would like the instrument to beep upon reaching the target temperature e Position the cursor over the Lid Control Mode step and tap the touch pad ONCE Tapping twice will select the step for editing which will be covered in a later chapter If you mistakenly select the step for editing and the Lid Control Mode window appears just select lt Cancel gt at the bottom of the window Once the step is selected any new steps that are added will be inserted after the Lid Control Mode step Tip Before entering a new step always select the insertion point first e Select lt Temp gt Temperature Step Ez Step 1 Temperature 7 c Beep on Target Hrs Min Sec Time rT Tl l Forever Options Increment Temp Set Parametere Extend Time Set Parametere Slow Ramp Get Parametere 5 30 Creating Programs W The Temperature Step window presents the operator with a nu
55. chine that is excessively oily or that emits ionizing radiation upon arrival at our factory PLEASE ELIMINATE ALL BIOHAZARDS AND RADIA TION 3 Pack the machine in its original packaging If this has been misplaced or discarded call MJ Research to request shipment of packaging materials You can also request a loaner machine which will be provided if available a rental fee may apply You can use the loaner s packaging to return the machine needing repair Remove the Alpha unit from the DNA Engine Dyad base before shipping All warranties are voided if a machine is shipped with an Alpha unit installed If the Alpha unit also needs to be shipped pack it in its original packaging materials 4 Write the RMA number on the outside of the box 5 Ship the machine freight prepaid to the following address We recommend you pur chase insurance from your shipper Ship to Repair Department MJ Research Incorporated 590 Lincoln Street Waltham MA 02451 C 1 Appendix D Warranties E Appendix D Warranties U S amp Canadian Limited Warranty Standard MJ Research Incorporated warrants NEW MJ RESEARCH BRAND THERMAL CYCLERS MOD ELS PTC 100 PTC 150 PTC 200 PTC 220 amp PTC 225 against defects in material and workmanship for a period of two years from the date of purchase If a defect is discovered MJ Research will at its option repair replace or refund the purchase price of the THERMAL CYCLER at no charge to the custom
56. cientists Adding a forever incubation step is identical to programming a temperature step with the exception of selecting the lt Forever gt box after specifying the incubation temperature The duration of the step will be displayed as a blue infinity symbol Creating Programs W Program GRAPH E Lid Mode CONSTANT t00 0c Unused temperature step Add Step after Control Mode cac Temp Grad Golo Forever Step 5 forever temperature step Delete Step Save Save As Save Aun Cancel If there are any additional steps displayed in the graphical programming window that you do not wish to include in the program select the step and then select lt Delete Step gt We do not wish to include the temperature step displayed in the fifth temperature gradi ent step column Our completed program appears as follows Program GRAPHHI E Lid Mode CONSTANT amp 100 00 Add Step after Control Mode carc Temp Grad Golo M Forever SavetHun Cancel Select lt Save gt to save the completed program GRAPH 1 Note If your completed graphical program still bears the name TEMPLATE select lt Save As gt and enter an appropriate name for the program see the Using the File Save As Window section in this chapter A graphical program with the default name TEMPLATE can not be run 5 27 M DNA Engine Dyad Operations Manual 5 28 Entering a Program Using Advanced Mode Select l
57. cing protocol into a Dyad program Finally we will use a cycle sequencing example to illustrate the programming process step by step Front Panel Setup The various components of the Dyad control panel see figure 2 2 enable the operator to enter navigate and manipulate programs These programs are necessary to control the various dynamic capabilities of the Dyad cycler Note Chapter 4 covers the basic operation of the DNA Engine Dyad cycler Please read Chapter 4 for a complete description of the control panel and power up procedures Let s review The control panel components include Display Screen This is a 1 4 VGA display screen approximately 10cm x 12 5cm located at the left side of the control panel It displays all Dyad cycler operating parameters and can be controlled by the cursor buttons touch pad or external mouse and the numeric keypad Caution Unlike the touch pad the display screen is not a touch screen and should not be used to enter programming items Please avoid touching the dis play screen Cursor Keys These are four cursor keys located to the right of the display screen They can be used to navigate through various menu and selection items The use of these keys is optional as ALL screen selections can be done using the touch pad or the external mouse device The up down keys are primarily used to scroll vertically through various submenus and to toggle through selection options in a list T
58. cle field and select 5 35 M DNA Engine Dyad Operations Manual 5 36 Enter 5 in the field Select lt OK gt This program step incorporating a per cycle increase in incubation time would appear as follows Incubate at 60 C for 00 01 00 Increase by 5 0 seconds every cycle This step is not included in our cycle sequencing protocol so we continue with our next addition Entering a Goto Step As currently entered our program will run one cycle and then end What we really want it to do is run 30 cycles total This involves the insertion of a goto step Goto steps are useful for cycling your commands a predetermined number of times We have decided to cycle the steps in ADV 1 twenty nine more times Select lt GoTo gt Golo Step E Step 4 Go to step number Additional Number of Cycles OF Cancel The GoTo Step window appears Position the cursor in the Goto step number field and select Enter 2 in the field Note We do not want to include step one in the cycling process as this is our initial denaturation step and should not be repeated more than once Position the cursor in the Additional Number of Cycles field and select Enter 29 in the field Select lt OK gt Creating Programs W The program now appears as follows Temperature Control Mode Calculated Lid Control Mode Constant at 100 C l 2 4 Incubate at 94 C for 00 01 00 Beep on Target Incubate at 92 C f
59. completed 3 Slow Ramp Temperature modification which allows for slower temperature ramping than the default maximum rate of 3 0 C sec The minimum rate cur rently allowed is 0 1 C sec Slower ramp times than this may be achieved using a combination of increment and goto steps Contact MJR Technical Support at 888 652 9253 for details 4 Beep Modifies a temperature step or ramp step so the instrument will beep when the target temperature is reached 5 5 M DNA Engine Dyad Operations Manual Types of Programs There are two types of Dyad programs graphical and advanced Graphical programming features a graphical interface and a graphical representation of the program steps Ad vanced programming features a textbased interface and a descriptive listing of the program steps Graphical program Advanced program Program GRAPHH E Lid Mode constant 100 0 Program ADYH1 Lid Control Mode Constant at 100 C 1 Incubate at 94 0 C for 00 01 00 2 Incubate at 92 0 C for 00 00 30 Gradient from 45 C to 65 C for 00 03 00 4 Cycle to step 2 for 29 more times 5 Incubate at 10 0 C forever End El Add Step after Bi Control Mode carc Add Step After Temp Gradient Golo Lid Temp Grad Golo M Forever Save Hun Cancel Delete Step Save Run Save Save As Cancel Graphical Programs Creating a graphical program is desirable if you prefer the graphical programming interface and
60. continue 7 3 E DNA Engine Dyad Operations Manual 7 4 Running Programs Note The Operations Chapter Chapter 4 provides information on running programs with respect to the operation and use of Alpha units For example a program containing a gradient step will only run in 96 well Alpha units Please review this chapter for additional technical detail to ensure that the program you load and run is an appropriate match for your Alpha units To run a program displayed in either the advanced programming window or the graphical programming window select lt Save Run gt Recall that prior to running a newly created graphical program the program must be assigned a name other than the default name TEMPLATE by performing a save as Graphical programs with the default name TEMPLATE can not be run The Run Program window will appear allowing you to specify the block on which the proto col should be run and the calculated control parameters for the run if applicable as de scribed below To run previously created programs see Creating Programs Chapter 5 and Managing and Editing Programs Chapter 6 you must first load them into memory From the Status window e Select lt Run gt at the bottom of the window or from the lt Command gt menu e From the open program window that appears select the desired Folder and Program Double check the program listing to ensure that the listed steps are consistent with the
61. d snap the lid closed Make sure that the lid from the original probe tube fits the new tube tightly The probe wire may touch the sides of the tube The thermistor should rest on the bottom of the tube Caution The thermistor is extremely fragile Handle it with great care Adding the Oil Viscous oils are the best choice for the probe tube s representative sample They closely mimic the thermal characteristics of buffer solution which changes temperature sluggishly due to the high specific heat of water MJ Research recommends using heavy mineral oil for the following reasons e The calculations required to determine the correct volume of oil are easy e It is widely available and inexpensive Add mineral oil to the probe tube in the following proportions 1X the volume of the buffer in an individual tube plus 1X the volume of oil overlay if one is used It is important to use the correct amount of oil so that the representative sample changes tem perature at the same rate as the actual samples To add the oil open the sample tube and pipette in the appropriate amount The oil must completely cover the thermistor Light and heavy silicone oil may also be used but necessitate more complex calculations to determine the amount to add to the probe tube See Appendix 4 C of this chapter for infor mation on using these oils Note Use only mineral oil or silicone oil as the representative sample Do not use paraffin wax or Chill out
62. d to Calculated control gr been achieved as quickly as expected This may indicate a possible problem with the Alpha unit The instrument has detected unexpected temperature readings This may indicate a problem with the Alpha unit A ground fault was detected in an Alpha unit A problem was detected in a Power Bonnet lid Troubleshooting I Action It you place a 96 well Alpha unit in this quadrant you may run this gradient program No action required Temperature mode has been automatically switched to Calculated control for this run only Call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada eplace fuse on instrument and confirm that instrument is functional If problem reoccurs please call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or or Canada or your local distributor outside the US or or Canada or your local distributor outside the US or 10 7 M DNA Engine Dyad Operations Manual Problems in Power Up Should a Dyad cycler not power up properly as indicated in Chapter 4 please follow the steps outlined in the flowchart figure 10 1 to determine the best course of action In this case a problem should be consistent and repeatable Figure 10 1 Power up Troubleshooting Flowchart Tum power on gt Plug cycler into n ain 220Vac lt _ _ s unit plugged
63. desired program e Select lt OK gt Running Programs E You are now presented with the Run Program window The Run Program window Program MYFIRST Non GUEST Select Blocks Select All Clear 7 Indicates Block Type Used for Calc Mode OF Cancel The Run Program window allows you to select the Block s that you wish to run your protocol on You can select a single block or all blocks based on block compatibility with your program If you are preparing to run a program using calculated temperature control selecting lt OK gt will display a Select Calculated Mode Parameters window that is appropriate for the type of program and Alpha unit that you are using Parameters entered here will allow precise temperature calculations by the Dyad cycler for your specific protocol and Alpha unit For example for a 96 well Alpha unit the following selection window will appear Select Calculated Mode Parameters Reaction Yolume ul Enter a value between 10 pl and 100 ul C Polycarbonate Plates M Polypropylene Plates and Tubes OF Cancel e Enter the reaction volume of your samples and select the type of reaction 7 5 E DNA Engine Dyad Operations Manual vessels used e Select lt OK gt to initiate the run Refer to the Running Multiple Programs section at the end of this chapter for information on simultaneously running multiple programs During the Run Run Status Informati
64. e scenarios for programming this initial temperature step 1 If there are no steps displayed Select lt Temp gt temperature step will be added as the first step in the protocol with a default temperature of 55 0 C and a duration of 30 seconds Proceed to scenario 3 for instructions on editing step parameters 2 If the first step displayed is a gradient step Select the gradient step step 1 Select lt Temp gt This will add a new temperature step to the protocol as step 2 with a default temperature of 55 0 C and a duration of 30 seconds Note Steps are always added after the step that is currently selected Select the gradient step step 1 Select lt Delete Step gt The initial gradient step will be deleted and the newly added temperature step will be promoted to step one Proceed to scenario 3 for instructions on editing step parameters M DNA Engine Dyad Operations Manual 5 22 3 If the first step displayed is a temperature step or if a temperature step was added as indicated in scenarios 1 or 2 e Position the cursor text pointer in the temperature field of step 1 and select by tapping clicking once A flashing insertion point will appear in the temperature field See the Selecting a Step and Editing Step Parameters sections on page 5 20 for additional selection and editing options e Use the backspace key on the numeric keypad to delete the cur rent temperature if it is not 9
65. e vents as needed Remove light collections of dust with a softbristle brush or damp cloth Severe collections of dust and debris should be vacuumed out Turn the instrument off prior to cleaning or vacuuming air vents Use a solid non perforated support material when using the Dyad cycler on a wire rack Ensuring That Air Is Cool Enough Do not position two or more DNA Engine Dyad cyclers or other thermal cyclers so that the hot exhaust air of one blows directly into the air intake vents of another Make sure the DNA Engine Dyad cycler receives air that is 31 C or cooler by measuring the temperature of air entering the machine through its air intake vents Place the DNA Engine Dyad cycler where you plan to use it and turn it on Try to reproduce what will be typical operating conditions for the machine in that location particularly any heat producing factors e g nearby equipment running window blinds open lights on Run a typical protocol for 30 minutes to warm up the DNA Engine Dyad cycler then measure the air temperature at the back air intake vents If more than one machine is involved measure the air temperature for each If the air intake temperature of any machine is warmer than 31 C use Table 3 1 to troubleshoot the problem Some experimentation may be required to determine the best solution when more than one cause is involved After taking steps to solve the problem verify that the temperature of the air entering the air
66. e Dyad Operations Manual 10 14 Alpha Units and the Remote Alpha Dock System Alpha Units Available from MJ Research Inc 11 2 About the Remote Alpha Dock System 11 4 Packing checklist 11 5 Requirements 11 5 Environment 11 5 Power Supply 11 6 Air Supply 11 7 Installation 11 7 Operation 11 8 m DNA Engine Dyad Operations Manual Alpha Units Available from MJ Research Inc Alpha unit interchangeable sample block heat pump assemblies are available in a pal ette of different configurations to accommodate a wide variety of thermal cycling applica tions All Aloha units are compatible with any of the cycler bases from the DNA Engine line including the DNA Engine Dyad and Tetrad bases All Alpha units deliver the same thermal profiles with the same NIST traceable accuracy no matter what instrument they are plugged into and swapping an Alpha unit takes just ten seconds Available Alpha units include The 60 for 0 5ml Tubes ALS 1260 This block holds sixty O 5ml microfuge tubes Many researchers prefer this format because the tubes are easy to use economical and each is large enough to write on The 96 for 0 2ml Tubes or 96 well Plates ALS 1296 This sample format can hold either one 96 well microplate or 96 x O 2ml tubes or strips of O 2ml tubes These V well vessels are specifically designed for thermal cycling and they have become the industry standard A thermal gradient rangin
67. e US or Canada or your local distributor outside the US or Canada Call 888 MJ CYCLE in the US or Canada or your local pe distributor outside the US or overheating the has Canadal been automatical An irregu arity 23 21 or 20 i No action required at this time you will be notified if any oroblem is found sensors in the heat sink and HS PS SF PT power supply To prevent Troubleshooting I Remote Error Code Reason Action Error Code S Call 888 MJ CYCLE in the U or Canada or your local distributor outside the US or Canada The internal fan is not providing adequate cooling for the instrument An irregularity ha detected in a heated lid sensor The heated lid has been temporarily turned off The No action required at this time sensor will be checked and you will be notified if any problem is found Call 888 MJ CYCLE in the US detected in the sample block or Canada or your local There may be a problem with a distributor outside the US or Peltier module A memory fault has been found Stored may be affected or Canada or your local distributor outside the US or rograms or call 888 MJ CYCLE in the ooe memory has been DS FC du o your local distributor outside the US or f posa be Th IN Check probe and connections e In sample probe Ihe If problem reoccurs please call thermal control method has 888 MJ CYCLE in the US or st lo TEAN
68. e appropriate insertion point if not high lighted already and select Note The appropriate insertion point would be selected by highlighting the first step Insertion will occur AFTER this step 5 31 M DNA Engine Dyad Operations Manual 5 32 Select lt Temp gt Position the cursor over the Temperature field and select Enter 92 from the numeric keypad into the field Similarly enter 30 in the Time Sec field Additionally we want the cycler to beep after reaching the target temperature Select Beep on Target At this point in the Temperature Step window all selections have been made according to our protocol Select lt OK gt In the advanced programming window the program listing now appears as follows Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target 2 Incubate at 92 C for 00 00 30 Beep on Target End The 92 C denaturation is the first step in the cycling portion of our program The next step is the gradient step Highlight Step 2 in the program listing Select lt Gradient gt Creating Programs W Gradient Step xjl Step 3 Lower Temperature C Higher Temperature C Hrs Min Sec Time J J I Forever Options Extend Time Set Parameters Preview OK Cancel Entering a Gradient Step The Gradient Step window indicates the steo number and includes fields for specifying the Lower Temperature and
69. e learned how to select our options based on the type of Alpha unit and sample vessels used in our experiment We have learned how to terminate a program run Finally we have learned how to use the most versatile feature of the Dyad cycler by running multiple program simulta neously In subsequent chapters we will discuss the remaining menu and submenu items particularly the Utilities 7 9 8 Using the Utilities lt About gt 8 2 lt User Name gt 8 3 lt Set Date Time gt 8 3 lt Remote gt 8 4 lt Auto Remote gt 8 4 lt Update Soft gt amp lt Network Config gt 8 5 lt Ping gt 8 7 Additional Utilities 8 8 Transferring Program Files 8 8 M DNA Engine Dyad Operations Manual In previous chapters you ve learned how to install and operate the Dyad cycler as well as write and execute programs In this chapter the various functions of the lt Utilities gt submenu will be discussed The lt Utilities gt submenu rounds out the capabilities of the Dyad cycler providing access to user name control remote command mode and the date and time settings The lt Utilities gt submenu is selectable from the Status window Here is a review of the available submenus lt About gt When this item is selected a screen like the following will appear Version 107 Dec i 2001 Va CLERTTILOR AL UE Peltier Thermal Cycler Front End Software Version 1 07 Dec 71 2001 Engine Software Versions Engine 1 31
70. e rejected and the default value or last valid value will reappear Deleting a Step To delete a step first select the step as indicated above such that the line or arrow depicting that step turns red Then select the lt Delete Step gt button The selected step will be deleted and the following step will automatically be promoted Adding a Step In graphical programs a step is added directly after the step that is currently se lected Graphical programs can contain a total of six temperature and gradient steps and one goto step To add a step select the step that will immediately proceed the new step Then select either the lt Temp gt lt Grad gt or lt Golo gt button to add either a temperature gradient or a goto step to the program A goto step can not be the first or only step in a program See the sections immediately following for complete in structions on adding specific types of steps and entering step parameters Now let s begin entering the steps for our example program Creating Programs W Entering a Temperature Step Recall again our raw program Use calculated temperature control mode Use constant lid control mode at 100 C An initial incubation at 94 C for 1 minute l 92 C for 30 seconds 2 Gradient from 45 C to 65 C for 3 minutes 3 Goto step 1 29 more times A An incubation at 10 C forever D END The first actual step in the protocol is the incubation at 94 C for 1 minute There are thre
71. ely shuts down and cannot be turned back on The machine records the event as a power loss If a protocol is running when a fuse blows the machine will resume the run when the fuse is replaced and the power restored J Warning The DNA Engine Dyad cycler incorporates neutral fusing which means that live power may still be available inside the unit even when a fuse has blown or been removed Never open the Dyad base You could receive a serious electrical shock Opening the base will also void your warranty 1 Disconnect the power cord from the back of the instrument Move the power switch to the O off position 2 Insert a small flat head screwdriver into the slot in the center of the fuse plug figure 9 1A and gently turn The plug will disengage Pull the plug straight out to expose the fuse figure 9 1B 3 Remove both fuses and replace them with new ones it is often impossible to determine visually which fuse is blown You can also test the fuses with an ohmmeter to determine which is defective and replace just that one 4 Gently press the fuse plug back into place Turn and secure with the screwdriver Recon nect the power cord Figure 9 1 A How to pull out the fuse plug B Location of the fuses in the opened plug Fuse plug 9 3 M DNA Engine Dyad Operations Manual 9 4 10 Troubleshooting Sources of Problems 10 2 System Problems 10 2 Error Messages 10 3 Problems in Power Up 10 8 Problems with
72. er provided the product is returned to MJ Research within the warranty period Refer to Appendix C for shipping instructions In no event will MJ Research be responsible for damage resulting from accident abuse misuses or inadequate packaging of returned goods and MJ Research disclaims all liability for consequential dam ages resulting from defects of any kind UNLESS OTHERWISE PROHIBITED BY LAW ALL IMPLIED WARRANTIES INCLUDING MER CHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE LIMITED IN DURATION TO TWO 2 YEARS FROM THE DATE OF ORIGINAL RETAIL PURCHASE OF THIS PROD UCT This warranty gives you specific legal rights You may also have other rights that vary from state to state Some states do not allow limitations on how long an implied warranty lasts so the above limitation may not apply to you The warranty and remedies set forth above are exclusive and in lieu of all others oral or written expressed or implied No MJ Research dealer agent or employee is authorized to make any modification addition or extension to this warranty except in the form of the extended warranty outlined below MJ Research is not responsible for special incidental or consequential damages resulting from any breach of warranty or under any other legal theory including downtime lost samples or experiments lost reagents lost profits goodwill damage to or replacement of equipment property and any costs of recovering or reproducing experimen
73. er 60 as an example and Time enter 30 seconds as an example e Select Slow Ramp e Select lt Set Parameters gt for the Slow Ramp option The Slow Ramp window appears M DNA Engine Dyad Operations Manual Slow Ramp Option E Ramp to 60 0 C C per second OF Cancel e Position the cursor over the C per second field and select e Enter 0 5 e Select lt OK gt You would expect the program step as included in a protocol to appear as fol lows Incubate at 60 0 C for 00 00 30 Ramp to 60 C at 0 5 C per second The Increment Temp option The Increment Temp option is useful for modifying a temperature step to allow a per cycle increase or decrease of temperature each time the step is executed see The Elements of a Program near the beginning of this chapter for more information Move from the programming window to the Temperature Step window using the lt Temp gt button as before Enter the appropriate incubation Temperature enter 60 as an ex ample and Time enter 30 seconds as an example e Select Increment Temp e Select lt Set Parameters gt for the Increment Temp option The Increment Temperature window appears Increment Temperature M Increase Decrease by C per cycle OF Cancel 5 38 Creating Programs W Select Decrease Position the cursor over the by _ per cycle field and select Enter 0 2 in the field Select lt OK gt You would expect
74. f 5 1 tracking mode 5 11 Lid control step advanced program 5 5 5 37 Loading sample vessels 4 10 M Maintenance 9 2 Microplates removing 3 6 selecting 4 7 Microseal A film 4 8 Microseal B adhesive seals 4 9 Microseal M sealing mat 4 9 Microseal P pads 4 14 Mode selection window 5 16 5 17 5 28 Monitoring the run 7 6 Mouse device connecting 3 2 selection switch 2 3 Mouse keys 2 2 4 3 See also Touch pad buttons Mouse port location of 2 3 Mouse touch pad selection switch location of 2 3 Moving programs 6 11 Multiple programs running 7 8 Index E In 5 M DNA Engine Dyad Operations Manual N Network config utility 8 5 8 6 Numeric keypad See also Control panel location 2 2 O Opening a program 6 2 6 4 advanced mode 6 2 6 3 basic mode 6 4 Operation turning the Dyad cycler on 4 2 P Packing checklist 3 2 Password utility 8 8 Pausing a run 7 7 7 8 Peltier effect B 1 Ping utility 8 7 8 8 Ports 4 4 ethernet 2 3 RS 232 2 3 8 4 Power Bonnet lid 2 4 Power cord location of jack 2 3 plugging in 3 2 Power supply requirements 3 3 acceptable power cords 3 3 Power switch location of 2 3 Probe adding representative sample calculating amount of oil to add 4 12 choosing type of oil 4 12 how to add oil 4 12 silicone oil as representative sample 4 17 connecting to block 4 12 customizing probe vessel 4 11 detecting faulty 4 13 function of 4 11 layout 4 11 loading into block 4 12 thermistor 4 11 4 12 P
75. fin waxes solidify at room temperature The wax can then be pierced with a micropipette and the samples drawn off from below the wax Silicone oil and mineral oil can be poured off or aspirated from tubes if the samples are first frozen 15 to 20 C The samples are usually pure enough for analysis without an extraction Chill out liquid wax available from MJ Research is an easy to use alternative to oil This purified paraffinic oil solidifies at 14 C and is liquid at room temperature By programming a hold at low temperature the wax can be solidified at the end of a run A pipette tip can then be used to pierce the wax in the tubes and remove the samples The wax is available in a clear optical assay grade or dyed red to assist in monitoring its use The red dye has no adverse effects on fluorescent gel analysis of reaction products Sealing with the Hot Bonnet Lid The Hot Bonnets heated inner lid maintains the air in the upper part of sample vessels at a higher temperature than the reaction mixture This prevents condensation of evaporated water vapor onto the vessel walls and lid so that solution concentrations are unchanged by thermal cycling The Hot Bonnet lid also exerts pressure on the tops of vessels loaded into the block helping to maintain a vapor tight seal and to firmly seat tubes or the plate in the block Caps film adhesive seals or mats must be used along with the Hot Bonnet lid to prevent evaporative losses Note W
76. g from 1 C up to 24 C can be programmed across this block allowing you to optimize reaction conditions in a single ex periment The 384 for 384 well Plates ALS 1238 MJ Research Inc has worked with several genome centers to develop a high density format for automated operation The result is the 384 well Alpha unit a true 4X version of the 96 well format The wells have the same V profile and are spaced on 4 5mm centers The Flat Block for Microarrays and Customized Attachments ALS 1200 384 well heated lid ALS 1201 96 well heated lid ALS 1203 no heated lid The Flat Block surface is ideal for microarrays and biochips and it provides the flexibility to customize our industry leading thermal cyclers for your specific needs The 30 30 Dual Block for 0 5ml Tubes ALD 1233 In many labs multiple users compete for time on a thermal cycler but rarely does a single user fill a 60 well block to capacity At other times an investigator may wish to run a single experiment with differing thermal parameters to optimize a protocol For such circumstances dual block Alpha units are available The blocks are independent these hold 30 x 0 5ml tubes and each has its own heated lid The 48 48 Dual Block for 0 2ml Tubes ALD 1 244 An alternative dual design has two blocks that hold 48 x 0 2ml tubes or a 48 well microplate Each independent block has an integral heated lid The O 2ml format works especially well in oil free
77. gh during the course of nor mal operation to cause burns or cause liquids to boil explosively Wear safety goggles or other eye protection at all times during operation The PTC 220 DNA Engine Dyad cycler incorporates neutral fusing which means that live power may still be available inside the machine even when a fuse has blown or been removed Never open the PTC 220 DNA Engine Dyad cycler base you could receive a serious electrical shock Opening the base will also void your warranty Never remove an Alpha unit from the PTC 220 DNA Engine Dyad cycler with the power turned on and a program running Doing so can cause electrical arcing that can melt the contacts in the connector joining the Alpha unit to the PTC 220 DNA Engine Dyad cycler Explanation of Symbols A AN L Identifies components that pose a risk of personal injury or damage to the instru ment if improperly handled Identifies components that pose a risk of electrical shock if improperly handled Identifies components that pose a risk of personal injury due to excessive heat if improperly handled A 1 M DNA Engine Dyad Systems Operations Manual A 2 Safe Use Guidelines The PTC 220 DNA Engine Dyad cycler is designed to be safe to operate under the following conditions Indoor use Altitude up to 3000m Ambient temperature 5 C 31 C Relative humidity 10 90 noncondensing Transient overvoltage per Installation Category Il IEC 664 P
78. gram modifications are available in advanced programs see the Types of Programs section immediately following for more information on ad vanced programs 1 Increment Temp Modifies a temperature step to allow a per cycle increase or decrease of temperature 0 1 C to 10 0 C per cycle each time the step is executed This feature is useful when annealing stringency is a consideration such as in a touchdown program In a touchdown program the annealing temperature begins higher than the cal culated temperature and incrementally decreases each cycle first reaching and eventually falling below the calculated annealing temperature With the reaction beginning at a temperature favoring high stringency in hybridization and incrementing to lower stringency the reaction favors the desired product by cre ating a high proportion of signal relative to noise in the early amplification cycles 2 Extend Time Modifies a temperature step to allow a per cycle lengthening or shortening of a temperature step hold by 1 60 sec cycle each time a step is executed This capability is useful for slowly increasing typically by 2 to 5 sec onds per cycle the hold time during an extension step The number of bases that a polymerase must synthesize during the extension step increases in later cycles because there are more template molecules because there are fewer active poly merase molecules or both The extra time can allow synthesis to be
79. hapters In the Status window the lt View gt submenu allows the user to view the lt Error Log gt Entries will be made to this log if a user terminates a program before its end if the cycler terminates a program before end or if the cycler encounters a serviceable issue before the termination of the program but was able to complete the program Problems and error messages can either be cycler or Alpha unit specific In each case a recommended action is listed and should be followed Under no circumstances should a customer attempt service of a Dyad cycler or an Alpha unit as this will result in a voided warranty and may not result in complete problem resolution The only exception would be minor maintenance of the units such as removal of vent blockage or routine cleaning as outlined in Chapter 9 Troubleshooting I Table 10 1 Error Messages The Error Codes listed in the table below correspond to the error codes returned in the Dyad cycler s lt Error Log gt The Remote Error Codes listed correspond to the error codes returned when the Dyad cycler is operated in remote mode see the lt Remote gt and lt Auto Remote gt sections in Chapter 8 for more information on remote mode Note The Dyad system software is quite sensitive to block and heat sink errors for these can affect accuracy and performance When such error messages occur try restarting the proto col If the message fails to reappear proceed as usual Error Code
80. he bottom of the window will list the total number of ping attempts the number and percentage of pings received from the host and the number and percentage of pings lost i e not received from the host 8 7 M DNA Engine Dyad Operations Manual PDL Sockets TCP IP Kernel Minimize button Close button select to exit do not select Select lt Ping gt to repeat the ping or select the minimize button to exit the window Note Do not exit this window by using the close button If you inadvertently exit using the close button an error will appear directing you to power cycle the instrument Additional Utilities The lt Password gt and lt Service gt utilities are intended for use by MUR personnel only Other functions may be added to the Dyad system software in future updates Please follow wWww mir com for update information Transferring Program Files During the initial boot up sequence of the Dyad cycler see the Turning the DNA Engine Dyad Cycler On section in Chapter 4 several options are available for transferring pro gram files Options 2 Send Files and 3 Receive Files can be used to establish a Dyad cycler to Dyad cycler transfer of all program files in all folders To perform a Dyad cycler to Dyad cycler file transfer 1 Use a standard DB9 nullmodem cable to connect the Dyad cyclers via their RS 232 ports 2 During the boot up sequence of the sending instrument select the Send Files option by ente
81. he left right keys are used to navigate through menu bars and to move through all available buttons or options in any given window Creating Programs W Numeric Keypad This is located to the right of the cursor buttons and consists of a typical numeric keypad numbers O through 9 and lt lt ENTER gt gt key There is also a backspace delete key and a decimal button The numeric keypad is used to enter parameters such as temperature hold time and cycle iterations Touch Pad Located on the right side of the control panel and bearing the MUR logo the touch pad is a touch sensitive mouse emulation device approximately 8cm x 6 4cm Essentially all pro gram maneuvers and navigation can be accomplished using the touch pad and the numeric keypad The touch pad is used to move a pointer that is visible on the display screen Fingertip movement on the touch pad will result in similar pointer movement Hence a fingertip dragged from left to right on the touch pad will result in movement of the pointer trom left to right on the display screen Once the pointer is positioned over a menu or selection item on the display screen a single tap of the touch pad with a fingertip will implement the command The only exception is during the editing of programming steps Selection of programming steps for editing re quires a double tap Tapping the touch pad has the same effect as the left touch pad button Y Tip When programming we recommend not
82. he program select lt Yes gt If you want to keep your newly created program select lt No gt 6 10 Managing and Editing Programs W Moving a Program e Select lt Programs gt in the Status window e Select lt Move gt from the drop down menu From Folder Hove Program To Folder ADVANCED BASIC EXAMPLE Moving is the same as copying described above with one distinction only one copy of the program is maintained in the To Folder The copy in the From Folder is deleted You will not be prompted with a verification step for this move so exercise some caution Deleting a Folder No command set would be complete without a folder maintenance window Folders must be empty before deletion e Select lt Programs gt in the Status window e Select lt Delete Folder gt from the drop down menu e To delete a folder highlight it and select lt Delete gt Delete Folder Delete Exit 6 11 E DNA Engine Dyad Operations Manual 6 12 You will be presented with a confirmation screen asking you to verify the dele tion Select lt Yes gt to delete the folder In this chapter we have utilized editing tools to add and subtract steps from our example programs In addition we learned to utilize file and folder tools to maintain our program lists In subsequent Chapters we will learn more about the menu bar of the Status window and how to navigate the utilities and functions located there We wil
83. hen tubes are cooled to below ambient temperatures a ring of condensation may form in tubes above the liquid level but below the top of the sample block This is not a cause for concern since it occurs only at the final cool down step when thermal cycling is com plete Microseal A film offers a quick alternative to sealing microplates or arrays of tube strips This film is specially designed to seal tightly during cycling yet release smoothly to minimize the risk of aerosol formation and cross contamination of samples Microseal A film is easily cut for use with fewer than 96 samples Operation W Microseal B adhesive seals feature an aggressive adhesive effective from 20 C to 110 C which allows secure sample storage or transport before and after cycling The clear polyes ter backing allows easy inspection of sample wells Microseal B clear adhesive seals are ideal for thermal cycling in all polypropylene and polystyrene microplates Microseal M rubber sealing mats are an economical means to seal 96 well microplates An array of 96 dimples on the mat helps orient it on the microplate and prevents the mat from sticking to the heated lid The mats may be cleaned with sodium hypochlorite bleach for reuse and they are autoclavable Adjusting the Hot Bonnet Lid s Pressure The pressure exerted by the Hot Bonnet lid must be manually adjusted to fit the sample vessels being used Once set the Hot Bonnet lid can be opened
84. horter than 1 hour 40 minutes and forever incubations are also allowed While all graphical programs can be opened and edited in advanced mode only a subset of advanced programs can be opened and edited in basic mode Advanced programs that meet the criteria outlined above for graphical programs can be opened in basic mode see Opening a Program in Chapter 6 for more information To create an advanced program refer to the Entering a Program Using Advanced Mode section below Designing a New Program The first step in designing any program is the translation of your experimental protocol into Dyad program steps We suggest writing all steps until you are reasonably comfortable with Dyad programming For purposes of this explanation we will be working with a cycle sequencing example First we will write down the raw steps then make some modifications with the parameters that were described in the previous section The Elements of a Program and then determine what our final program should be The actual implementation and entering of program steps will be covered in a later section Note You will soon become familiar with Dyad program design and be able to enter steps directly from experimental protocols However we strongly suggest following these steps the first few times through as they will probably save troubleshooting time later 5 7 M DNA Engine Dyad Operations Manual 5 8 Let s Start with an Example
85. ight Alpha units are In use Using the Data Ports The DNA Engine Dyad cycler has two data ports located at the rear of the machine an RS 232 port and an Ethernet port See Chapter 8 for information on using these ports Operating Alpha Units Note Operation of the Twin Towers unit will not be discussed owing to the many differences between this type of Alpha unit and the others Please see the Twin Towers Block Operations Manual for operating instructions Note Alpha units equipped with Power Bonnet lids are installed and removed as described below See the Power Bonnet Lid User s Manual and the Entering a Lid Control Step section in Chapter 5 for information on opening and closing Alpha units with Power Bonnet lids Installing an Alpha Unit Caution Do not insert or remove an Alpha unit with the DNA Engine Dyad cycler turned on electrical arcing can result Read the safety warning in Appen dix A regarding electrical safety when inserting or removing an Alpha unit 1 Turn the DNA Engine Dyad cycler off see the Caution above 2 Hold the Alpha unit at its front and back edges 3 Lower the Alpha unit into the DNA Engine Dyad base leaving at least 3cm between the front edge of the Alpha unit and the front of the base 4 Raise the handle at the back of the Alpha unit and slide the block forward as far as it will go see figure 4 1A 5 Push the handle down until it is completely vertical see figure 4 1B fi
86. indow Creating Programs W Program Untitled Lid Control Mode Tracking at 5 C above End a Add Step After Temp Gradient Golo Lid Delete Step Save Run Save Save As Cancel It is from the advanced programming window that you will add steps using the lt Temp gt lt Gradient gt lt Goto gt and lt Lid gt options Additionally buttons running across the window bottom provide options to lt Delete Step gt lt Save Run gt lt Save gt or lt Save As gt programs and lt Cancel gt the current programming ses sion The lt Save gt and lt Save As gt buttons are probably the most important buttons since a program that is saved can be used or edited at a later date Select lt Save As gt As the File Save As window is identical in both graphical and advanced pro gramming mode follow the instructions in the Using the File Save As Window section above to create a new folder named FOLDER2 and a new program named ADV 1 After selecting lt OK gt you will be returned to the advanced programming win dow The following steps will appear to indicate your progress with the program ADV 1 Temperature Control Mode Calculated Lid Control Mode Constant at 100 C End 5 29 M DNA Engine Dyad Operations Manual Entering a Temperature Step While the program ADV 1 is a bona fide Dyad program it has no utility A run of this program will finish immediately after its start be
87. ing samples to desired incubation temperature rapidly 5 9 M DNA Engine Dyad Operations Manual 5 10 2 Incubation periods are timed according to how long the samples not the blocks reside at the target temperature 3 The instrument automatically compensates for vessel type and reaction volume Note We will choose calculated control for our example Block Control The Dyad cycler maintains the block at the programmed temperature independent of sample temperature This mode of temperature control is common to older models of thermal cyclers Block control provides less accurate control of sample temperatures than calculated con trol provides Under block control the temperature of samples will lag behind the tem perature of the block The length of the time lag depends on the vessel type and sample volume but typically is between 10 and 30 seconds Block control is chiefly used to run protocols developed for other thermal cyclers that use block control or if you use the lt Instant gt command to incubate samples at a set temperature for long periods of time In Sample Probe Control The Dyad cycler adjusts the block s temperature to maintain the probe at programmed temperatures You may purchase an in sample probe from MJ Research Inc The in sample probe is comprised of a temperature probe placed inside a typical capped tube A thin cable exits the top and may be plugged into the Alpha unit Probe control is available
88. ing solution into the block s wells and then heat the block in an attempt to clean it Severe damage to the block the heated lid and the chassis may result Cleaning the Air Vents Clean the air intake and exhaust vents with a soft bristle brush a damp cloth or a vacuum cleaner whenever dust is visible in them The air intake vents are located on the bottom lower front edge and back of the machine the air exhaust vents are located on both sides see figures 2 1 2 3 and 2 4 If these vents become clogged with dust and debris airflow to the Alpha unit s heat sink is hampered causing performance problems related to over heating The air intake vents are particularly likely to collect dust since their holes are much smaller than those of the air exhaust vents to prevent debris from entering the instrument Maintenance Tip To prevent problems with overheating institute a regular program of checking for dust buildup particularly for robotics installations Cleaning Radioactive or Biohazardous Materials From the Block When cleaning machines that have been running radioactive or biohazardous reactions consult your institution s radiation safety officer or biosafety officer regarding cleaning meth ods monitoring and disposing of contaminated materials Changing the Fuses The circuits in the DNA Engine Dyad cycler are protected by two fuses 6 3A fastacting 5 x 20mm When a fuse blows the DNA Engine Dyad cycler immediat
89. intake vents has been lowered using the procedure outlined above Installation Table 3 1 Troubleshooting Air Supply Problems Cause Possible Remedies Air circulation is poor Provide more space around machine or adjust room ventilation Ambient air temperature Adjust air conditioning to lower ambient air temperature is high Machine is in warm part Move machine away from or protect machine from such heat of room sources as radiators heaters other equipment or bright sunlight Machines are crowded Arrange machines so that warm exhaust air does not enter intake vents Requirements for Robotics Installations Robotics installations require special attention to airflow and air temperature Typically in these installations DNA Engine Dyad cyclers and other thermal cyclers are restricted to a small area along with other heat generating equipment Overheating can quickly occur when many of these instruments are operating at once unless preventive measures are taken Follow the procedures described above to ensure adequate airflow and an air intake tem perature of 31 C or cooler Air intake temperature must be verified by measurement Do not use oil or glycerin to thermally couple sample vessels to the blocks of machines in a robotics installation This can make plates difficult to remove 3 5 M DNA Engine Dyad Operations Manual 384 Well Microplate Specifics Some users find that a 384 well microplate can be difficult
90. ion and purifi cation protocols Add additional purification steps if necessary Check nucleic acid preparations by gel electrophoresis Troubleshooting I Problems due to Environmental Conditions Setup and Maintenance Strict adherence to the installation operation and maintenance instructions provided in Chapters 3 4 and 9 are tantamount to the continued trouble free operation of the Dyad cycler Should it be determined that the source of a problem is due to incorrect operation or setup consult Table 10 3 for a list of problems and suggested solutions Table 10 3 Environmental difficulties Problem Cause Action Frequent shutdowns Machine is not receiving enough Make sure air intake vents are due to overheating air not obstructed by dust debris Frequent Slow Block or paper Remove light collec Cycling HS tions of dust and debris with Overheat and HS damp cloth Vacuum out heavy Overheating error collections Remove any papers messages placed under the machine Posi tion machine at least 10cm from vertical surfaces Air flowing into intake vents is Check temperature of air enter not lt 31 C ing air intake vents following procedure on p 3 4 If higher than 31 C use Table 3 1 to troubleshoot and remove cause s Dust and debris clog Failure to regularly check for Remove light collections with ging up air intake buildup damp cloth Vacuum out heavy vents collections 10 13 M DNA Engin
91. l also learn how to run our new program VA Running Programs Using the Instant Incubation Feature 7 2 Running Programs 7 4 The Run Program window 7 5 During the Run 7 6 Run Status 7 6 Terminating a Run 7 7 Pausing Resuming a Run 7 7 Skipping a Step 7 8 Inaccessible Features 7 8 Running Multiple Programs 7 8 E DNA Engine Dyad Operations Manual In Chapter 5 we translated an experimental protocol into functional Dyad programs In Chapter 6 we edited program steps In this chapter we explore the implementation of the Dyad programs and the instant incubation feature Using the Instant Incubation Feature The DNA Engine Dyad cycler can also be quite useful as an instant constanttemperature incubator with a range from 5 0 C to 105 C 4 C to 100 C with the Twin Towers block This feature can be used for performing ligations digestions etc or with slides overnight humidified hybridizations Note The Twin Towers block can be used as a humidified chamber for steady state incuba tions e g hybridizations color development reactions To humidify a block push one labo ratory tissue into the bottom slot and inject 1ml of deionized water onto it See the Twin Towers Operations Manual for complete instructions To initiate an instant incubation from the Dyad Status window Programs Command Tools View Utilities Block is Inactive 96 F User Name GUEST dk El Temperature Time Block 246
92. l become step 3 Managing and Editing Programs W Inserting a Step Now we will insert an annealing temperature step Highlight step 2 Recall that new steps are inserted AFTER the highlighted step Select lt Temp gt from the advanced programming window The Temperature Step window appears Select an incubation Temperature of 60 C a hold Time of 3 minutes and the Beep on Target option Enter parameters as described in Chapter 5 Your program should appear as follows Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target 2 Incubate at 92 C for 00 00 30 Beep on Target 3 Incubate 60 C for 00 03 00 Beep on Target 4 Cycle to step 2 for 29 more times 5 Incubate at 10 C forever End Editing a Step Step 2 of the program ADV 1 includes a 30 second incubation We wish to change that incubation to 25 seconds Select Step 2 by double clicking You are presented with the Temperature Step window The Temperature field shows 92 C and the Time field shows 30 seconds Select the Time Sec field Change the time from 30 to 25 Select lt OK gt 6 7 E DNA Engine Dyad Operations Manual Now the Program Editing window lists the program steps as the following Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target 2 Incubate at 92 C for 00 00 25 Beep on Target 3 Incubate 60
93. le program GRAPH 1 now appears as follows 6 5 E DNA Engine Dyad Operations Manual Program GR4PH41 E Lid Mode CONSTANT 100 0 Add Step after Bi Control Mode carc Temp Grad Golo M Forever Delete Step Save Save As Save Run Cancel Editing an Advanced Program Highlighting and Selecting Program Steps Several important factors determine step selection The insertion point for new steps is AFTER the step highlighted in the program listing This highlighting is accomplished with a SINGLE tap or click Double tapping clicking on a program step will immediately open the appropri ate step editing window for the highlighted step The windows for editing are the same as those for step creation Selection can also be done via the left touch pad button In order to insert or edit steps the Dyad user should become familiar with these conventions By default in this manual we use the single touch pad tap mouse click to highlight a step and a double tap click to select it Deleting a Step The lt Delete Step gt button will allow us to delete a program step To begin the process of changing step 3 of our advanced program ADV 1 into a temperature step 6 6 Highlight step 3 by positioning the pointer over the first line of step three and tapping once Select lt Delete Step gt The gradient step will be deleted and the following steps renumbered For ex ample the go to step previously step 4 wil
94. lecting a Step When a step is selected the line s or arrow depicting that step will turn red There are several ways to select a step You can select a step by positioning the cursor in the step s column and tapping or clicking once A flashing insertion point will appear in the temperature field of that step To select a specific temperature or time field or a goto step position the cursor its shape will change to a text pointer in the desired tield and tap click once to display an insertion point or tap click twice to highlight the entire field Alternatively use the left right cursor keys to sequentially select the temperature time and or number of cycles fields for the program steps Editing Step Parameters If you have selected a step such that a flashing insertion point appears in the field you wish to edit use the backspace and number keys on the numeric keypad to first delete the current value then enter the desired temperature time or number of cycles Tap the touch pad or click the mouse once to accept the change or press the left right cursor key once to accept the change and move to the next field If you have selected a step such that the field you wish to edit is highlighted use the number keys to enter the desired temperature time or number of cycles Tap the touch pad or click the mouse once to accept the changes If an inappropriate value is entered such as an incubation temperature of 110 C the change will b
95. ll blocks including the Block Name Block Status the name of the Program Running the Time Remaining in the program the Time Elapsed and the User name select the lt View gt menu and then lt Status All gt trom the drop down list Status All Blocks EJ Block Block Program Time Time Name Status Running Remaining Elapsed User 1 96 Active ADYH 02 10 34 00 06 14 GUEST 296 Active ADYHI 02 10 34 00 06 14 GUEST Completed with Mo Errors Completed with Errors If the Block Status indicates that the program was completed with errors select lt Error Log gt from the lt View gt menu to view error messages To view a run log including the date and time that a program was initiated by a user select lt Cycler Log gt from the lt View gt menu Terminating a Run To terminate a run prior to completion select the appropriate block from the block selection menu in the Status window and select lt Stop gt Alternatively select the lt Command gt menu in the Status window and then select lt Stop gt and either lt All gt to terminate all programs run ning on all blocks or select the desired block from the drop down list Pausing Resuming a Run To merely pause a run select the appropriate block from the block selection menu in the Status window and select lt Pause gt Alternatively select the lt Command gt menu in the Status window and then select lt Pause Resume gt and either lt Pause All gt to pause all programs running o
96. lling Shipping instructions C 1 Skipping a step 7 8 Slow ramp option 5 37 5 38 Software update 8 5 8 6 Software version 8 2 Specifications DNA Engine Dyad cycler 2 5 Gradient 2 5 See also Gradient Status 7 6 7 7 block cycle and lid temperatures 7 6 time elapsed 7 7 time remaining 7 7 Status all 7 7 Status window 5 14 8 2 Syntax checking 8 4 Index E In 9 M DNA Engine Dyad Operations Manual T Temperature control methods block control 5 10 calculated control 5 9 choice of calculated control over probe control 4 11 in sample probe control 5 10 modifying a program from a different machine 5 11 modifying block and probe control programs 5 10 Temperature hold time 5 12 Temperature increment See Increment temp option Temperature range of Dyad cycler 2 5 Temperature step advanced program 5 30 5 33 graphical program 5 21 Temperature step window 5 31 Terminating a run 7 7 Thermal accuracy 2 5 Thermal uniformity 2 5 Thermoelectric unit B 1 Time Date setting 8 3 Touch pad See Control panel Touch pad buttons See Control panel Touch pad location of 2 2 Touch pad mouse selection switch location of 2 3 Touchdown 5 5 See also Increment temp option Tracking mode 5 11 Transferring program files 8 8 Dyad cycler to Dyad cycler 8 8 Troubleshooting error messages 10 3 problems in power up 10 8 problems w environment setup or maintenance 10 13 problems w system performance 10 9 problems with alpha unit 10 10 protocol proble
97. location of 2 3 Index E In 3 M DNA Engine Dyad Operations Manual G Goto step 5 5 5 8 advanced program 5 36 5 37 graphical program 5 25 5 26 Gradient step 5 5 advanced program 5 33 5 35 graphical program 5 24 5 25 Gradient using to optimize 5 12 Gradients gradient preview window 5 34 specifications 2 5 accuracy 2 5 calculator accuracy 2 5 column uniformity 2 5 lowest highest temperature 2 5 temperature differential range 2 5 Graphical program adding a step 5 20 deleting a step 5 20 editing 6 5 6 6 See also Editing a program editing a step 5 20 forever incubation entering 5 26 goto step entering 5 25 5 26 gradient step entering 5 24 5 25 opening 6 4 selecting a step 5 20 specifications 5 6 temperature step entering 5 2 5 24 Graphical programming window 5 1 9 5 20 adding a step 5 20 deleting a step 5 20 editing a step 5 20 forever incubation entering 5 26 goto step entering 5 25 5 26 gradient step entering 5 24 5 25 selecting a step 5 20 temperature step entering 5 2 5 24 Graphs window 5 14 H Hold time 5 12 Hot Bonnet lid adjusting lid pressure 4 9 4 13 In 4 In sample probe control 5 10 Increment temp option 5 5 5 38 5 39 Inserting a step advanced program 6 7 Inserting a step graphical program 5 20 6 5 Instant incubation 7 2 K Keyboard virtual 5 18 Keypad 5 3 L Layout back view 2 3 bottom view 2 3 control panel 2 2 front view 2 2 Lid control methods constant mode 5 1 of
98. mber of tempera ture adjustment options The Temperature and Time fields as well as the Beep on Target selection option are available at the top of the window At the bottom the Increment Temp Extend Time and Slow Ramp options are available each with their own lt Set Parameters gt button For this particular step the only parameters that will be set will be the temperature and the incubation time Note The Alpha units can ramp at a maximum of 3 0 C per second If a slower ramp speed is not entered this will be the default Position the cursor over the Temperature field and select Enter 94 from the numeric keypad into the field Similarly enter 1 in the Time Min field Additionally we want the cycler to beep after reaching the target temperature Select Beep on Target At this point in the Temperature Step window all selections have been made according to our protocol Select lt OK gt In the advanced programming window the program listing now appears as follows Temperature Control Mode Calculated Lid Control Mode Constant at 100 C 1 Incubate at 94 C for 00 01 00 Beep on Target End The program if run now would ramp to 94 C for 1 minute and then end Let s continue to enter steps and build our program The first temperature step in the cycling portion of our program incubates at 92 C for 30 seconds We would also like the cycler to beep after reaching this tem perature Position the cursor over th
99. more times Incubate at 10 C forever End Managing and Editing Programs W After running the above program analysis of the resulting sequencing data indi cated that 60 C was the best annealing temperature We would like to change step 3 from a gradient step into a temperature step Further step 2 includes a 30 second incubation that we wish to change to 25 seconds Editing a Graphical Program As graphical programming discussed in Chapter 5 involves essentially editing a TEMPLATE program we will only briefly discuss the specifics of editing a preexisting graphical program here Please refer to Entering a Program Using Graphical Mode in Chapter 5 specifically The Graphical Programming Window section for more information To replace step 3 the gradient step with a temperature incubation at 60 C first open the program GRAPH 1 in basic mode as described above e Select the gradient step e Select lt Delete Step gt e Select step 2 the temperature incubation at 92 C Recall that new steps are added AFTER the selected step e Select lt Temp gt The gradient step has now been replaced with a default temperature step Follow the instructions in Chapter 5 for entering temperature step parameters To decrease the incubation time of step 2 from 30 seconds to 25 seconds e Select the time field of step 2 e Delete 30 and enter 25 using the numeric keypad e Tap or click once to accept the change Our examp
100. ms 10 10 system problems 10 2 U Uniformity See Thermal uniformity Update soft utility 8 5 8 6 User name 8 3 Utilities about 8 2 auto remote 8 4 network config 8 5 8 6 password 8 8 ping 8 7 remote 8 4 In 10 Index E service 8 8 set date time 8 3 update soft 8 5 8 6 username 8 3 V Virtual keyboard 5 18 W Warranties D 1 In 11 M DNA Engine Dyad Operations Manual In 12 Declaration of Conformity AXA m Research tnc DECLARATION OF CONFORMITY for attachment of CE mark as well as to document Canadian amp US compliance T rir tn Dai n Jriginotc of Feifier lhermal y MJ RESEARCH INCORPORATED manufacturer of the DNA Engine Dyad Peltier thermal cycler Model PTC 220 hereby declares this equipment conforms to the following APPLICATION OF E U Council DIRECTIVES 73 23 EEC 89 336 EEC 93 68 EEC STANDARDS TO WHICH CONFORMITY IS DECLARED EU IEC 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part including Amendments 1 amp 2 EU EN61326 1997 Annex B amp A1 1998 Electrical Equipment for Measure ment Control and Laboratory Use EMC Requirements including Radiated amp Conducted Emissions and Immunity CANADA CAN CSA C22 2 No 1010 1 92 amp No 1010 1B 97 Safety Require ments for Electrical Equipment for Measurement Control and Laboratory Use Part including Amndmnts 1 amp 2 CSA Cert 1125786 LR 97357 10
101. mum programmable temperature is 30 C Note The programmable temperature gradient feature is only accessible if a 96 well Al pha unit s is mounted in the Dyad cycler The gradient feature is not compatible with other types of Alpha units The temperature of any well or column in the sample block may be displayed using the lt Gradient Calculator gt available from the lt Tools gt drop down menu in the Status window Since our oligonucleotide annealing temperature is not optimized we will replace our an nealing step with a gradient step We will optimize in the range of 45 C to 65 C Creating Programs W Your written program should now appear as follows Raw program Use calculated temperature control mode Use constant lid control mode at 100 C l 92 C for 30 seconds 2 Gradient from 45 C to 65 C for 3 minutes 3 Goto step 1 29 more times 4 END Beyond the Example Protocol Other Considerations In addition to the above considerations you can also include other protocol variations which will further optimize the yield and quality of your product For example an initial extended denaturation step can serve to destroy any heat labile nucleases and other potentially interfering components while ensuring that the nucleic acid has been completely denatured and prepped for annealing In some protocols after the final elongation step a slow temperature ramp can also be included to ensure proper product annealing
102. n all blocks or select the desired block from the drop down list 7 7 E DNA Engine Dyad Operations Manual 7 8 To resume a run select lt Resume gt from the bottom of the Status window or select lt Com mand gt lt Pause Resume gt and either lt Resume All gt or select the desired block from the drop down list Skipping a Step To skip to the next step in a running program select lt Skip gt at the bottom of the Status window A confirmation screen will ask you to confirm the skip select lt Yes gt to skip to the next step in the protocol displayed in the Status window Inaccessible Features Considerations must also be made for the compatibility of the program with the installed Alpha units The Run Program window will display one or more grayed out Alpha units in the Select Blocks section if there is an incompatibility with the program in queue or if an Alpha unit is currently unavailable due to a protocol that is already running Some scenarios which may be the cause of inaccessible features or display changes include Block control A sample temperature will not be displayed Probe control Sample temperature will be replaced with Probe temperature Additionally programs with lid temperature steps will not run with probe control Gradient step Alpha units other than 96 well Alpha units will be grayed out Lid step Alpha units will be grayed out if a lid step is included in the protocol but there
103. nced program 6 7 Adding a step graphical program 5 20 6 5 Advanced mode programming 5 28 5 39 Advanced program deleting a step 6 6 editing 6 6 6 8 See also Editing a program editing a step 6 7 6 8 extend time option 5 35 5 36 goto step entering 5 36 5 37 gradient step entering 5 33 5 35 increment temp option 5 38 5 39 inserting a step 6 7 lid control step entering 5 37 opening 6 2 6 3 slow ramp option 5 37 specifications 5 7 temperature step entering 5 30 5 33 Advanced programming window 5 29 Air exhaust vents location of 2 2 Air intake vents location of 2 2 2 3 Air supply requirements ensuring adequate air supply 3 4 ensuring air is cool enough 3 4 troubleshooting problems with 3 4 Alpha units closing 4 6 dual block models 2 4 installing 4 4 opening 4 6 removing 4 5 single block models 2 4 slide block 2 4 Auto remote utility 8 4 In 1 M DNA Engine Dyad Operations Manual Basic mode programming 5 15 5 39 Bleach using in block 9 2 Block control 5 10 Block selection incompatibility 7 8 Block status lights 4 4 C Calculated control 5 9 Chill out liquid wax 4 8 Cleaning 9 2 air vents 9 2 biohazardous materials 9 3 chassis and block 4 16 9 2 radioactive materials 4 16 9 3 removing oil from block 9 2 solutions to use 4 16 Condensation in tubes following holds 4 8 Constant mode 5 11 Constanttemperature incubator use as 7 2 Contrast adjustment knob location of 2 3 Control panel 2 2 4 3 5 2 block sta
104. nd results we recom mend use of an MJ Research Cleanbox or equivalent which utilizes a UV lightsource to inactivate extraneous DNA Three models of cleanbox are offered by MJ Research CBX 0750 75cm wide single door 120V CBX 0900 90cm wide single door 120V CBX 0120 120cm wide dual doors 120V To clean block wells use swabs moistened with water 95 ethanol or a 1 100 dilution of bleach in water see the Twin Towers Operations Manual for instructions on cleaning the Twin Towers slide slots If using bleach swab wells with water afterward to remove all traces of bleach Clean spilled liquids out of the block as soon as possible dried fluids can be difficult to remove Do not clean the block with caustic or strongly alkaline solutions e g strong soaps ammonia bleach at a higher concentration than specified above These will damage the block s protective coating possibly causing electrical shorting If you use oil in the block a practice not recommended by MJ Research Inc see Using Oil to Thermally Couple Sample Vessels to the Block in Chapter 4 clean the wells whenever the oil has become discolored or contains particulate matter Use a swab to determine whether cleaning is needed Clean the block with 95 ethanol as described above Oil buildup must be prevented Old oil harbors dirt which interferes with vessel seating and diminishes thermal coupling of sample vessels to the block Caution Do not pour any clean
105. nded outlet Figure 11 3 Attachment of power and data cables Dock connector Remote Alpha Dock base Alpha Units and the Remote Alpha Dock System E Air Supply e Alpha units being operated in the remote configuration have no operating constraints that do not also apply to normal operations in the PTC 220 Installation e Turn the dock connector upside down so that the green circuit board is visible Check that the ground lead remains attached at both ends fig 1 1 1 Note the two female multi pin sockets one labeled DATA and the other POWER e Connect the multi pin power cable s male end to the female socket labeled POWER and slide the latch to lock the pins in place e Both of the data cable s multi pin connectors are male one is labeled CONNECTOR DATA and the other DOCK DATA Attach the CONNECTOR DATA end to the fe male connector labeled DATA on the circuit board and slide the latch to lock the pins in place e Press both cables firmly into the two strain relief holes on the Dock Connector s front side fig 11 2 e Turn the Remote Dock upside down You will see a male multi pin connector labeled Power Port and a female multi pin connector labeled Data Port fig 11 3 Figure 11 4 Fan power supply connected in series D TN DU 11 7 m DNA Engine Dyad Operations Manual
106. nly Temperature gradient available with the 96 well Alpha unit only Outside of the example protocol other considerations Choosing a Temperature Control Mode The Dyad cycler can control incubation temperature in three possible ways each of which has different implications for the speed and accuracy of sample heating These include Calculated Control Block Control and In Sample Probe Control Calculated Control When using calculated control the Dyad cycler estimates sample temperatures based on the block s thermal profile the rate of heat transfer through the sample tube or slide and the sample volume or mass Since this estimate is based on known quantities and the laws of thermodynamics sample temperatures are controlled much more accurately than with block or probe control Since the sample temperature will always lag behind the block temperature the Dyad cycler can adjust the block temperature to bring samples of a specific volume in a spe cific vessel type to programmed temperatures This is done through optimized over shoots of the block temperature by a few degrees for a few seconds which bring samples to the desired temperature more quickly Calculated control is also the method of choice for most types of programs because it yields the most consistency reliability and speed Calculated control provides for shorter protocols in three ways 1 Brief and precise block temperature overshoots are used to br
107. nymous with click on point and click and any phraseol ogy implying selection of menu or option items with a mouse Particularly with the Dyad cycler select should symbolize any physical selection on the Dyad input devices touch pad mouse numeric keypad cursor keys This includes single or double taps on the touch pad pressing of the left touch pad button or pressing the lt lt Enter gt gt button on the numeric keypad The Elements of a Program Dyad programs consist of a combination or series of steps and setup parameters that repre sent protocol requirements Note The procedures involved in actually entering these steps will be described in subse quent pages but please familiarize yourself with the types of steps used to create Dyad programs The considerations behind choosing various elements will be explained further in the Con siderations During Program Creation section The following is a summary of the individual program elements and their basic functions Temperature Control Mode This parameter defines the temperature control al gorithm used during the program run The three different modes include Calcu lated Block and In Sample Probe Due to the expected lag of sample tempera ture behind block temperature the Dyad cycler can use calculated mode to compen sate accordingly The Dyad cycler defaults to Calculated Refer to the Choosing a Temperature Control Mode section below for additional information
108. ollution degree 2 in accordance with IEC 664 Electromagnetic Interference The PTC 220 DNA Engine Dyad cycler has been tested and found to comply with the limits for a Class A digital device pursuant to part 15 of the US FCC Rules These limits are designed to provide a reasonable protection against harmful interference when the equip ment is operated in a commercial environment These machines generate use and can radiate radiofrequency energy and if not installed and used in accordance with the instruc tion manual may cause harmful interference to radio communications Operation of these machines in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense In addition the PTC 220 DNA Engine Dyad thermal cycler designs have been tested and found to comply with the EMC standards for emissions and susceptibility established by the European Union at time of manufacture Further the PTC 220 DNA Engine Dyad thermal cycler does not exceed the Class A limits for radio noise emissions from digital apparatus set out in the Radio Interference Regulations of the Canadian Department of Communications LE PRESENT APPAREIL NUMERIQUE N EMET PAS DE BRUITS RADIOELECTRIQUES DEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES DE CLASS A PRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIOELECTRIQUE EDICTE PAR LE MINISTERE DES COMMUNICATIONS DU CANADA
109. om web www mijr com NATIONAL SALES Orrice 987 Tahoe Boulevard 106 Incline Village NV 89451 USA Manuractory 590 Lincoln Street Waltham MA 02451 USA printed on 100 recycled paper DoC 1
110. ommended for use with the DNA Engine Dyad cycler and are available from MJ Research To place an order call 888 729 2165 or fax 888 729 2166 Key Y Reaction vessel fits block sealing option fits reaction vessel without modification 2 Reaction vessel sealing option can be cut to fit MJ Research Thermal Reaction Vessels Sealing Options for Oil Free Cycling Cycler Blocks 48 Description MJ Research Microseal Microseal Microseal 8 Strip 12 Strip Chill out A film B seals M mat caps caps wax a Catalog 454 5001 MSB 1001 MSM 1001 TCS 0801 15 1201 CHO series ae y Microseal skirted MSP series P y 384 well microplates ae Microseal skirted MSP series J p 96 well microplates TM Hard Shell skirted HSP series z V 96 well microplates Multiplate unskirted MLP series J V 96 well microplates MLL series Tee Multiplate unskirted Me ee V y 48 well microplates ae Multiplate unskirted MLP 2401 j y 24 well microplates PRE ENT Multiplate unskirted MLP 2501 J y 25 well microplates e e v e Microseal P pad MSP 1001 Concord skirted CON 9601 96 well microplates 8 strip 0 2 ml tubes TBS series TLS series 12 strip 0 2 ml tubes 0 2 ml tubes 0 2 ml tubes w caps TFI 0201 TWI 0201 0 5 ml tubes w caps Note Concord microplates are made from polycarbonate plastic which is more prone to poor sealing and vapor leakage during stringent thermal cycling Y Y Y Y Y Y
111. on in the Temperature Time and Cycle fields of the Status window will indicate that your program is running To graphically display the run conditions go to the Graphs win dow e Select lt Graphs gt at the bottom of the window or from the lt View gt menu To graph the Block Temp Sample Temp and or Lid Temp over time select the appropriate options near the bottom of the Graphs window The estimated Time Remaining in the program and the amount of Time Elapsed since the program was initiated are also indicated Program ADYi1 Active Block 1 96 User Hame GUEST Cycle 4 of 30 Time Remaining 01 57 30 Time Elapsed 00 16 59 M Block Temp M Sample Temp FLid Temp Run Instant Stop Pause Skip Status e Select lt Status gt to return to the Status window The Status window will display Block Sample and Lid temperatures correlating to the program running on the block chosen in the block selection menu These temperatures represent real time readings and correspond to the values repre sented graphically in the above window The time remaining in the current Step and the Remaining time in the program are displayed along with the current Cycle number 7 6 Running Programs E Tip Recall that convenient and rapid toggling between the Status and Graphs windows can be achieved by selecting the button in the lower right of the Status and Graphs windows respectively To simultaneously view the status of a
112. or 00 00 30 Beep on Target Gradient from 45 C to 65 C for 00 03 00 Cycle to step 2 for 29 more times End Now the program will run with 30 cycles As discussed previously it may be desirable in some cases to ramp to a tempera ture at a slower than maximum rate or to include an incremental increase or decrease in temperature per cycle Some operators may also wish to include instructions in a program to open and close a Power Bonnet motorized lid A Lid step and the Slow Ramp and Increment Temp options are not necessary in our example but the steps necessary to implement them are described below should they be needed in other protocols Entering a Lid Control Step When using a single block Alpha unit fitted with a Power Bonnet motorized lid it may be desirable to include steps in the Dyad program that direct the lid to open or close This can be particularly useful in robotic installations To include a lid control step in a program Select lt Lid gt in the programming window Select either Open Lid or Close Lid in the Lid Control window that appears The Slow Ramp Option Earlier we described the Extend Time option for a temperature step now we will select the Slow Ramp option see the Choosing a Temperature Ramping Rate section for additional information Move from the programming window to the Temperature Step window using the lt Temp gt button as before Enter the appropriate incubation Tempera ture ent
113. or wish to quickly enter a program that e Does not contain more than a total of six temperature and or gradient steps e Does not contain temperature or gradient steps that contain modifications i e increment temp extend time slow ramp beep e Does not contain more than one goto step e Does not contain a temperature incubation below 0 C e Does not contain a step with an incubation lasting more than 1 hour 39 minutes and 59 seconds 99 59 Forever incubations excepted In addition to the speed with which they can be entered another benefit of graphical pro grams is that they can be quickly edited This can be particularly useful if you tend to repeat edly run the same general protocol with limited changes e g varying annealing tempera tures To create a graphical program refer to the Entering a Program Using Graphical Mode section below 5 6 Creating Programs W Advanced Programs Advanced programs offer all of the Dyad programming features with the exception of the graphical programming interface Creating an advanced program is desirable if you wish to enter a program that e Contains many steps e Contains temperature or gradient steps with modifying instructions i e increment temp extend time slow ramp beep e Contains multiple goto steps e Contains a temperature incubation in the range of 1 C to 5 C e Contains an incubation lasting between 1 hour 40 minutes and 18 hours incubation times s
114. orever incubation Choosing A Thermal Gradient Molecular biology labs routinely optimize annealing and denaturing temperatures for ther mal cycling reactions Optimization is critical but not always easy The Tm melting tem perature of an oligonucleotide can be estimated using an empirically derived correlation which considers a combination of DNA length G C content and salt concentration How ever since the Tm is only an estimate the true annealing temperature may need adjusting in the actual experiment This optimization involves repeating a reaction at several different annealing temperatures which requires a great deal of time and monopolizes the instrument while several experiments are run in tandem To complicate matters further similar time consuming experiments may also be required for denaturing temperature optimization The Dyad cycler programmable temperature gradient feature allows for optimization of an incubation temperature in a single experiment by analyzing a number of different tempera tures simultaneously The thermal gradient delivers a controlled thermal difference left to right across the sample block This will result in a precisely defined temperature gradient that is repeatable from experiment to experiment The range of temperatures that can be achieved from left to right across a 96 well Alpha unit can be as small as 1 C or as great as 24 C The maximum programmable temperature is 105 C the mini
115. ou call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada An irregularity was detected in a power supply temperature sensor The sensor will be No action required at this time checked and you will be notified if any problem is found Please observe the following guidelines 1 place instruments at least 10cm apart 2 avoid lacing instruments in any location where hot air might The block took slightly longer enter the intake vents 3 place than expected to achieve the instrument on a hard surface with programmed temperature This AY pel i Ee de SBC CAF condition may be caused by c Er E duel T gs inadequate airflow or a unit fins of dust and debris 5 problem with ihe Alpha anit avoid running the Instrument in sell areas with ambient temperatures above 25 C Continue to use the instrument If problem reoccurs call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada igntly Call 888 MJ CYCLE in the US longer than expected to reach Canadal or id SLC ee ok side d distributor outside the US or sensor or lid heater Canada Remote Reason A program containing a gradient step can only be run on a 96 well Alpha unit The program entereo n run in Calculated or Block temperature control mode Probe mode is unavailable for this type of program For optimum results the temperature control mode has been automatically switche
116. ough the walls of O 2ml tubes of several different brands during thermal cycling No data are yet available on radioactive contamination with polypropylene microplates 0 5ml Polypropylene Tubes Contamination problems are rarer with this type of tube but instances have been re ported The Solution 1 Substitute the low energy beta emitter P in cycle sequencing 3P nucleotides are not subject to the same kind of chemical breakdown as 35S nucleotides and they have not been associated with volatile breakdown products m DNA Engine Dyad Operations Manual 2 If 5S must be used three things will help control contamination an oil overlay inside the tubes mineral oil in the thermal cycler outside the tubes and use of thick walled O 5ml tubes Always run 35S thermal cycling reactions in a fume hood and be aware that vessels may be contaminated on the outside after thermal cycling Please be certain that you are using the appropriate detection methods and cleaning procedures for this iso tope Consult your radiation safety officer for his or her recommendations If mild cleaning agents do not remove radioactivity harsher cleaners may be used occa sionally and carefully Users have suggested the detergent PCC 54 Pierce Chemical Co Rockford Illinois Pierce Eurochemie B V Holland Micro Cleaning Solution Cole Parmer Niles Illinois and Dow Bathroom Cleaner available in supermarkets Caution Harsh cleaning agents such a
117. plete reaction mix minus sample with a control template and primer set Use only high quality tubes plates that fit block snugly Ensure that wells are free of foreign materials that would interfere with tube plate seating 10 11 M DNA Engine Dyad Operations Manual Table 10 2 Protocol Difficulty Error Message Reaction is working but broad low mole cular weight band is seen in gels Reaction working but unexpected extra products or smear is seen 10 12 Cause and Result Primer dimer material often produces a broad band in the lt 100bp region of gels Nonspecific hybridization occur ring during setup Reaction component concentration too high or too low Annealing temperature too low Protocol contains a wrong value Template not of sufficient purity Multiple templates or host DNA in sequencing reactions Action If obtaining appropriate reac tion product s no need to change anything Minimize primer dimer production by designing primers with no 3 self complementarity Reoptimize magnesium concen tration and annealing temper ature to maximize desired pro duct and minimize primer dimers Program a hot start into the protocol Check concentratons of compo nents May need to reoptimize magnesium concentration Reoptimize annealing temperature Use List utility to check proto col s temperature control method temperatures and times Check extract
118. pment to do PCR is encouraged to contact Hoffmann La Roche for more information at one of the addresses below In the United States In other nations Licensing Manager PCR Licensing Manager Roche Molecular Systems Inc F Hoffmann La Roche Ltd 1145 Atlantic Avenue Building 222 350 Alameda CA 94501 USA CH 4002 Basel Switzerland 510 814 2970 41 61 687 3031 Fax 510 814 2977 Fax 41 61 687 2113 Contents Documentation Conventions cccccccccccccccccccccccececuucecceeuccsececeneusseetenenuseesecs iv Part The DNA Engine Dyad Thermal Cycler La Kore Keli e emer ner eter ner ett a nee A ne en net een E A l 1 1 LGVOU GANGS b GIIEQIONS A datant 2 FUSION OR asso dy teense manatee a atic wesnivesarescahaareateu aie cabenneniasre 3 1 Deraan eee en ee ere ene nen Ne ne eee Ne een et en ee 4 1 CTS GING TO GROIN S syns case a D do a ni de 5 1 Managing and Editing Programs na ne thin 6 1 R NMINO M LOGE CIN S needs uma 7 1 Orie Re OS id 8 1 PACING Ne NN ett EOE EEE E E E nn 9 1 OUR ES RO STARS ee eee teeta eater eer nn mney tere Vert nee a Rae 10 1 Part Il Accessories Alpha Units and the Remote Alpha Dock System 11 1 Appendix A Safety Warnings and Guidelines A 1 Appendix B How a Peltier Heat Pump Works cccceccceeeeseeeeeeeeseeeeeeenans B 1 Appendix C Shipping Instructions for US Residents ccccccceeeseeeeeeeeeees C 1
119. pward to open the lid The Hot Bonnet lid will tip backward revealing the entire block Caution Do not pull on the thumbwheel to open the unit This can damage the Hot Bonnet lid s mechanism Closing an Alpha Unit Press down on the top lever The lever will close down over the thumbwheel as the lid closes down over the sample block A click signifies that the Hot Bonnet lid s latch has engaged Selecting the Correct Sample Vessel The DNA Engine Dyad cycler s wide variety of interchangeable Alpha units affords great scope in choosing sample vessels Keep in mind that differences in tube and plate composi tion and wall thickness among the many brands available can affect reaction performance Protocols may require some adjustment to ensure optimum performance when using a new vessel type MJ Research offers a full range of tubes and microplates manufactured to the specifications of each type of Alpha unit to ensure a precise fit See Appendix 4 A of this chapter for a complete list Figure 4 2 Opening an Alpha unit Operation W 0 5ml Tubes Thick walled 0 5ml tubes may not fit tightly in thermal cycler wells and typically provide poor thermal transfer since these tubes were originally designed for centrifuges For best results we recommend using thin walled O 5ml tubes specifically designed for thermal cycling The higher quality brands provide a good and consistent fit MJ Research thin walled 0 5ml tubes are designed fo
120. r precise block fit and tight sealing of reactions down to 1Oul Thin Walled vs Thick Walled Tubes The thickness of sample tubes directly affects the soeed of sample heating and thus the amount of time required for incubations Thick walled tubes delay sample heating since heat transfers more slowly through the tubes walls For the earliest types of thermal cyclers this delay mattered little These machines ramping rates were so slow below 1 C sec that there was plenty of time for heat to transfer through the tube wall to the sample during a given incubation Modern thermal cyclers have much faster ramping rates up to 2 3 C second so the faster heat transfer provided by thin walled tubes allows protocols to be significantly shortened 0 2ml Tubes All types of thin walled O 2ml tubes may be used MJ Research offers high quality 0 2ml tubes in a number of styles including individual and strip tubes Microplates A variety of polycarbonate or polypropylene microplates can be used in Alpha units as long as they fit the wells snugly Polypropylene microplates are usually preferred because they exhibit very low protein binding and unlike polycarbonate microplates do not lose water vapor through the vessel walls This allows smaller sample volumes to be used as little as 5 1Oul Several varieties of microplates are available from MJ Research see the Tube Microplate and Sealing Selection Chart including Hard Shell
121. rd and attach a different connector Use a one piece molded connector If required additional dedicated power cords may be purchased through MJ Research or authorized distributors 3 3 E DNA Engine Dyad Operations Manual 3 4 Air Supply Requirements The DNA Engine Dyad cycler requires a constant supply of air that is 31 C or cooler in order to remove heat from the Alpha unit s heat sink Air is taken in from vents at the bottom and sides of the machine and exhausted from vents on both sides see figures 2 1 2 3 and 2 4 If the air supply is inadequate or too warm the machine can overheat causing performance problems software error messages particularly HS Overheating and Slow Block Cy cling and even automatic shutdowns Special attention should be paid to airflow and air temperature in robotics installations of DNA Engine Dyad cyclers Ensuring an Adequate Air Supply Do not block the air intake vents Position the DNA Engine Dyad cycler at least 10cm from vertical surfaces and other thermal cyclers greater distances may be required see below Do not put loose papers bench paper or this manual under the instrument they can be sucked into the air intake vents on the bottom Do not allow dust or debris to collect in the air intake vents The bottom air vents are particularly liable to collect dust and debris sometimes com pletely clogging up Check for dust and debris every few months and clean the intak
122. re 11 2 Remote Alpha Dock base and dock connector bottom view m DNA Engine Dyad Operations Manual Remote Alpha Dock bases with mounted Alpha units may be configured in any horizon tal orientation or array as long as a minimum side clearance of 10 cm is maintained between the Remote Alpha Dock unit and any wall bulkhead or adjacent unit this is identical to the PTC 220 base requirement Requirements for motorized lid operation or for loading or unloading plates may dictate additional clearances Remote Alpha Dock bases with mounted Alpha units may be stacked vertically as long as a minimum bottom clearance is maintained that would be no less than that resulting from the unit being placed on a solid horizontal platform A minimum top clearance is also required to allow access to and operation of the Alpha unit lid Remote Alpha Dock units can be flush mounted i e with the feet removed to facilitate robotic operation as long as the airway beneath the unit is equivalent to the airway the unit would have with the feet attached Usually a hole will need to be cut to allow air to flow to the cooling fan Figure 11 5 is a template for flush mounting the Remote Alpha Dock unit Power Supply The Alpha unit mounted in each Remote Alpha Dock base is powered from the PTC 220 base The Remote Alpha Dock unit s fan is powered externally and a power supply is pro vided that requires power from 90 250 VAC and 47 to 63 Hz with a grou
123. re using Turning the power off resets the base allowing it to recog nize the new block If not reset the base unit assumes that the previous type of block is installed resulting in error messages and procedural faults Alpha Units and the Remote Alpha Dock System E Figure 11 5 Flush mounting template 23 70 0 9 65 rly 2 84 14 Q a 168 20 6 6 D 165 00 Los D25 00 TYPICAL 1 0 DIMENSIONS IN MILLIMETERS AND IN NCHES di m DNA Engine Dyad Operations Manual 11 10 Appendix A Appendix A Safety Warnings and Guidelines W Safety Warnings and Guidelines AN Caution When removing an Alpha unit from a PTC 220 DNA Engine Dyad base keep all fingers and foreign objects away from the Alpha unit bays Keep all objects clear of the Alpha unit bays until the fan has come to rest Operating the PTC 220 DNA Engine Dyad cycler before reading this manual can constitute a personal injury hazard Only qualified labora tory personnel trained in the safe use of electrical equipment should oper ate these machines Do not open or attempt to repair the PTC 220 DNA Engine Dyad cycler base any Alpha unit or any accessory to the Dyad cycler Doing so will void your warranties and can put you at risk for electrical shock Return the PTC 220 DNA Engine Dyad cycler to the factory US customers or an authorized distributor all other customers if repairs are needed All Alpha unit blocks can become hot enou
124. reen port and push the connector into place 4 Pull the small switch located behind the purple port into the forward position see figure 2 4 The rear position will activate only the touch pad The forward position will activate only the external mouse device All Dyad cyclers are shipped with the touch pad enabled i e the switch is in the rear position Please note that the cycler will recognize either the touch pad OR a mouse but not both input devices simultaneously 5 Tip the Dyad cycler back down and power up the system Environmental Requirements Ensure that the area where the DNA Engine Dyad cycler is installed meets the following conditions for reasons of safety and performance e Nonexplosive environment e Normal air pressure altitude below 3000m e Ambient temperature 5 31 C e Relative humidity of 10 90 noncondensing e Unobstructed access to air that is 31 C or cooler see below e Protection from excessive heat and accidental spills Do not place the Dyad cycler near such heat sources as radiators and protect it from danger of having water or other fluids splashed on it which can cause shorting of its electrical circuits Power Supply Requirements The DNA Engine Dyad cycler requires 200 240VAC 50 60Hz and a grounded outlet on a minimum 20A line The Dyad cycler can use voltage in the specified range without adjust ment so there is no voltage setting switch Note Do not cut the supplied power co
125. ring 2 on the numeric keypad 3 During the boot up sequence of the receiving instrument select the Receive Files option by entering 3 on the numeric keypad The instrument will indicate that it is Ready to accept files 8 8 Using the Utilities Hi 4 To execute the file transfer select Proceed amp Exit when completed by entering 1 on the numeric keypad of the sending instrument Once the transfer is complete the TRANSFER COMPLETE message will be displayed and the instrument will proceed with the boot sequence 8 9 M DNA Engine Dyad Operations Manual 8 10 9 Maintenance Cleaning the DNA Engine Dyad Cycler 9 2 Cleaning the Chassis and Block 9 2 Cleaning the Air Vents 9 2 Cleaning Radioactive or Biohazardous Materials Out of the Block 9 3 Changing the Fuses 9 3 M DNA Engine Dyad Operations Manual 9 2 Cleaning the DNA Engine Dyad Cycler Cleaning the Chassis and Blocks Clean the outside of an Alpha unit or the cycler chassis with a damp soft cloth or tissue whenever something has been spilled on it or when the chassis is dusty A mild soap solution may be used if needed Allowing major buildup of laboratory dust or other contaminants may affect the performance of the cycler or Alpha units as well as the outcome of your experiments As with any thermal cycling experiment a reasonably clean contaminant free environment is recommended For particularly sensitive reactions where contamination could confou
126. rm pressure may be required but do not force the handle into position A definite click signals that the Alpha unit s connectors have mated with the DNA Engine Dyad cycler s connectors When the handle is in the down position the Alpha unit is locked into place Operation W Removing an Alpha Unit Caution Do not insert or remove an Alpha unit with the DNA Engine Dyad cycler turned on electrical arcing can result Read the safety warning in Appen dix A regarding electrical safety when inserting or removing an Alpha unit 1 Turn the DNA Engine Dyad cycler off see the Caution above 2 Pull upward on the handle When the lock releases you will hear a click and the Alpha unit will slide a little toward the back of the DNA Engine Dyad cycler The electrical connectors of the Alpha unit and the DNA Engine Dyad cycler are now disengaged so there is little danger of electrical shock 3 Slide the Alpha unit toward the rear of the DNA Engine Dyad cycler about 3cm 4 Grasp the front and back edges of the Alpha unit and lift it out of the machine Figure 4 1 Installing an Alpha unit 4 5 m DNA Engine Dyad Operations Manual 4 6 Opening an Alpha Unit Grip the front edge of the top lever of the Hot Bonnet lid as shown in figure 4 2A and pull upward firmly The top lever will pop open to reveal the entire thumbwheel see figure 4 2B Continue pulling u
127. robe control 4 11 5 10 In 6 Index E Program advanced 5 28 5 39 extend time option 5 35 goto step entering 5 36 5 37 gradient step entering 5 33 5 35 increment temp option 5 38 5 39 lid control step entering 5 37 slow ramp option 5 37 5 38 temperature step entering 5 30 5 33 advanced options beep on target 5 5 extend time 5 5 increment temp 5 5 slow ramp 5 5 copying 6 9 deleting 6 10 designing choosing temperature control method 5 9 choosing lid control method 5 1 choosing temperature gradient 5 12 See also Gradients choosing temperature hold time 5 12 choosing temperature ramping rate 5 11 example 5 8 other considerations 5 13 editing 6 4 6 8 See also Editing a program advanced 6 6 6 8 graphical 6 5 elements end step 5 5 go to step 5 5 gradient step 5 5 lid control mode 5 4 temperature control mode 5 4 temperature step 5 5 graphical 5 15 5 27 adding a step 5 20 deleting a step 5 20 editing a step 5 20 forever incubation entering 5 26 goto step entering 5 25 5 26 gradient step entering 5 24 graphical programming window 5 15 selecting a step 5 20 temperature step entering 5 21 template 5 15 inaccessible features 7 8 In 7 M DNA Engine Dyad Operations Manual monitoring the run graph window 7 6 status window 7 7 moving 6 11 opening 6 2 6 4 advanced mode 6 2 6 3 basic mode 6 4 running 7 4 run program window 7 5 running multiple 7 8 saving 35 17 5 19 6 8 types 5 6 5 7 advanced 5 7 gr
128. s and reactions see Current Protocols in Molecular Biology F Ausubel et al eds John Wiley amp Sons 10 10 Problem No reaction products obtained Cause Wrong protocol used Protocol contains a wrong value Reaction component omitted from mixture Denaturation temperature too low Annealing temperature too high for primers Wrong temperature control method used Probe failed causing machine to run protocol under calculated control Probe not filled with correct amount of oil Reaction mix contains an inhibitor e s heme from blood Reaction vessels not making good thermal contact with sample block Troubleshooting I Action Re run reaction using correct protocol Use List utility to check proto col s temperature control meth od temperatures and times Check reaction assembly proto col ensuring that mixture con tains appropriate components in correct concentrations Use 92 C for denaturation Only rarely are temps higher than 94 C required however Check for appropriate anneal ing temperatures of primers using available computer pro erams or empirical testing Use List utility to check temperature control method for protocol change if needed Check screen for probe failure error message Probe may need servicing or replacing Call MJ Research Inc or your local distributor Fill probe tube with correct amount of oil see p 4 12 Test a com
129. s those above are corrosive and must be thoroughly rinsed away within a few minutes of application They can eat away the surface finish of the blocks 4 16 Operation W Appendix 4 C Using Silicone Oil in the Probe Tube The following light and heavy silicone oils may be used instead of mineral oil as the repre sentative sample in a probe tube e Dow Corning 200 light silicone oil dimethypolysiloxane Sigma DMPS 5X Density 0 97g ml Viscosity 50cs Volume to use 1 7 x volume of buffer in individual sample tube plus one volume of oil overlay e Dow Corning 200 heavy silicone oil dimethypolysiloxane Sigma DMPS V Density 0 97g ml Viscosity 5cs Volume to use 2 7 x volume of buffer in individual sample tube plus one volume of oil overlay Note Use these oils only in the proportions outlined above Using them in any other proportion for example 1 1 with sample tube volumes will lead to inaccurate sample heating E DNA Engine Dyad Operations Manual 4 18 Front Panel Setup 5 2 Display Screen 5 2 Cursor Keys 5 2 Numeric Keypad 5 3 Touch Pad 5 3 Touch Pad Buttons 5 3 Programming Conventions 5 4 The Elements of a Program 5 4 Types of Programs 5 6 Graphical Programs 5 6 Advanced Programs 5 7 Designing a New Program 5 7 Let s Start with an Example 5 8 The Goto Option 5 8 Considerations During Program Creation 5 9 Choosing a Temperature Control Mode 5 9 Calculated Control 5 9 Block
130. started 10 3 M DNA Engine Dyad Operations Manual Error Code Please observe the following guidelines 1 place instruments at least 10cm apart 2 avoid lacing instruments in any pret where hot air might The heat sink temperature is enter the intake vents 3 place registering somewhat higher instrument on a hard surface with than normal Power as will no debris or paper underneath be scaled back slightly until the 4 clean all air vents and Alpha heat sink temperatue returns to unit fins of dust and debris 5 normal Adequate airflow is avoid running the instrument in necessary to prevent the heat areas with ambient temperatures sink from overheating above 25 C Continue to use the instrument If problem reoccurs please call 888 MJ CYCLE in the US or Canada or your local distributor outside the US or Canada Please observe the following guidelines 1 place instruments at least 10cm apart 2 avoid lacing instruments in any nerd where hot air might The heat sink reached a enter the intake vents 3 place temperature higher than instrument on a hard surface with normal and the program was no debris or paper underneath automatically stopped A clean all air vents and Alpha Adequate airflow is necessary to unit fins of dust and debris 5 prevent the heat sink from avoid running the instrument in overheating areas with ambient temperatures above 25 C If the problem reoccurs please call 888 MJ CYCLE in th
131. t With the machine switched off mount the Alpha units see the Installing an Alpha unit section in Chapter 4 Caution Do not insert or remove an Alpha unit with the Dyad cycler turned on electrical arcing can result Read the safety warning in Appendix A re garding electrical safety when inserting or removing an Alpha unit Optional External Mouse Device Included with each shipment of a DNA Engine Dyad thermal cycler is an externally attach able mouse intended to substitute for the function of the touch pad Should a Dyad user prefer an externally attached mouse device rather than the integrated touch pad the mouse should be attached prior to power up of the Dyad cycler Underneath the front lip of the Dyad cycler positioned at the mid point of the touch pad are two connection ports see figure 2 4 The purple port on the left is reserved for future function and should not be used The green port on the right is for connecting the external mouse To insure complete compliance with FCC and EMC requirements only a mouse with a ferrite core should be used with the Dyad instrument Installation W To connect the mouse please follow these steps 1 Verify that the Dyad cycler is off Wait for 10 seconds to ensure that all fans have stopped rotating 2 Grasping the sides of the Dyad cycler tilt the instrument back so that the underside of the lip is visible 3 Line up the pins of the mouse connector with the g
132. t Programs gt Note As described earlier this involves positioning the screen cursor over lt Pro grams gt with a fingertip on the touch pad and tapping the touch pad once Drop down submenus appear including lt Open gt lt New gt lt Copy gt lt Move gt lt Delete gt lt Delete Folder gt and lt New Folder gt Select lt New gt An additional menu appears allowing you to choose lt Advanced Mode gt or lt Basic Mode gt Select lt Advanced Mode gt You are now presented with the Mode Selection window It is at this point that you will choose the Temperature Control Mode and Lid Control Mode for the program Refer to the Considerations During Program Creation section earlier in this chapter for information on temperature and lid control modes Mode Selection Temperature Control Mode M Calculated Block In Sample Probe Lid Control Mode Constant M Tracking Set Parameters DK Cancel We have decided for the purposes of this example to use Calculated for our temperature control mode and Constant for our lid control mode Refer to the Using the Mode Selection Window section above for instructions on entering these choices into our advanced mode program The Mode Selection window is identical in both graphical and advanced programming modes However after selecting lt OK gt to accept any changes and exit the Mode Selection window you will return to the advanced programming w
133. tal results and data LIMITATIONS AND RESTRICTIONS This warranty applies only to machines sold in the U S A and Canada Under no circumstance will MJ Research ship a repaired or replaced machine or grant a refund of purchase price to a user in a nation in which there was an authorized MJ Research distributor at the time of purchase THIS WARRANTY IS NOT TRANSFERABLE FROM THE ORIGINAL PURCHASER TO A SUBSEQUENT OWNER FURTHERMORE THIS WARRANTY DOES NOT APPLY TO INSTRUMENTS USED OUTSIDE THE U S A OR CANADA EXCEPT WHEN EXPRESSLY AUTHORIZED IN WRITING BY MJ RESEARCH All provisions of this warranty are voided if the product is resold repaired or modified by anyone other than MJ Research or an authorized distributor D 1 M DNA Engine Dyad Systems Operations Manual D 2 U S amp Canadian Extended Warranty Optional MJ Research Incorporated will offer to each ORIGINAL PURCHASER of an MJ Research BRAND thermal cycler the opportunity to purchase an extension of the warranty coverage explained above for an additional two years The coverage must be purchased through a purchase order received by MJ Research within 30 days of receipt of the offer of extended warranty or the offer to renew the extended warranty These offers apply only to machines sold and used in the U S A and Canada Index E Index A About utility 8 2 Accessories See Power Bonnet lid Remote Alpha Dock system Accuracy See Thermal accuracy Adding a step adva
134. the back of the instrument see figure 2 3 Turn the power switch on The fan will turn on the display screen will illuminate and the microprocessor will implement a boot up protocol lasting about 10 seconds During the boot sequence the user is presented with several options including 1 Selftest choose the number 1 on the numeric keypad to instruct the Dyad cycler to perform a diagnostic system test and report any errors 2 Send Files choose the number 2 on the numeric keypad to prepare the Dyad cycler to transfer stored program files to another Dyad cycler see Chapter 8 for instructions on transferring program files 3 Receive Files choose the number 3 on the numeric keypad to prepare the Dyad cycler to receive stored program files from another Dyad cycler see Chapter 8 for instructions on transferring program files If no option is selected the boot sequence will automatically exit after approximately six seconds Following bootup the Dyad logo screen is briefly displayed The Dyad Status window will then be visible The DNA Engine Dyad cycler is now ready to accept edit and execute programs Programs Command Tools View Utilities Block is Inactive 96 F User Name GUEST eee I Temperature Time Block 248 C Sample 248 C Lid 23 4 C Cycle 0 Aun Instant Stop Pause GRIP Graphs Step 00 00 00 Remaining 00 00 00 Operation W Using the Control Panel The control panel see
135. thin wall microplates Hard Shell microplates feature a skirt and deck molded from a rigid thermostable polymer that com pletely resists the warping and shrinkage experienced with traditional one component plates The rigid skirt improves robotic handling such that stackers and robotic arms can grip and move Hard Shell plates securely and reliably In a separate step thin wall wells are molded of virgin polypropylene selected for low DNA binding and optimized for thermal cycling 4 7 E DNA Engine Dyad Operations Manual A 8 Sealing Sample Vessels To avoid changing the concentration of reactants steps must be taken to prevent the evapo ration of water from reaction mixtures during thermal cycling Only a layer of oil or wax will completely prevent evaporation from the surface of the reaction fluid However an ad equate degree of protection can be achieved by sealing vessels with caps film adhesive seals or mats then cycling the samples using the heated lid to prevent condensation Sealing with Oil or Wax Mineral oil silicone oil paraffin wax or Chillout 14 liquid wax may be used to seal samples Use only a small amount of oil or wax 1 3 drops 15 50ul are usually sufficient Include this volume in the total volume when setting up a calculated control protocol see Choosing a Temperature Control Mode in Chapter 5 Use the same amount of oil or wax in all sample vessels to ensure a uniform thermal profile Most paraf
136. to remove from the 384 well block after completing their thermal cycling protocol The plate fits very snugly in the block and the 384 points of contact can provide a significant amount of friction Fortunately it is relatively simple to ameliorate this problem if it occurs in your application In our experience a very thin coating of a Teflon based dry lubricant sprayed onto the block will solve the sticking problem very effectively The coating eventually wears off so the block should be re coated as needed probably about once every 10 to 20 runs Your experience will be the best guide in establishing the frequency for re coating As you will see a very thin coat is sufficient to eliminate any sticking TFE tetra fluoroethylene dry lubricant is available from many sources One source in the United States is Miller Stephenson Chemical Co Inc in Danbury CT 203 743 4447 in Morton Grove IL 847 966 2022 in Sylmar CA 818 896 4714 TFE Dry Lubricant Release Agent Cat MS 122DF aerosol 100z can approx 10 50 can Here are some guidelines for applying the TFE lubricant 1 Cool the block and lid to room temperature below 38 C 2 Cover the lid and any other areas that you don t want to get slippery 3 Shake the can well Spray for about 1 second onto the block 3 6 4 Operation Turning the DNA Engine Dyad Cycler On 4 2 Using the Control Panel 4 3 Display Screen 4 3 Operation Keys 4 3 Block St
137. to step This goto step can not be the first or only step in the protocol If there is a goto step blue arrow in the displayed protocol e Select the goto step e Select lt Delete Step gt To add a goto step to our protocol Select step 3 the last step to be included in the goto loop e Select lt GoTo gt A small red arrow will appear under step 3 M DNA Engine Dyad Operations Manual 5 26 e Select step 2 the first step to be included in the goto loop A red arrow will now extend from the end of step 3 to the beginning of step 2 and the default number of cycles the loop will execute 25 X will be displayed e Select the number of cycles and delete using the backspace key e Enter 29 from the numeric keypad and tap or click once to accept the change Program GRAFHAt E Lid Mode CONSTANT 100 00 Step 3 gradient step Step 4 goto step Add Step atter Control Mode carc Temp Grad D Forever Delete Step Save Save As Save Run Cancel Our program will now run with 30 cycles Entering a Forever Incubation Step 5 of our program is a forever incubation at 10 0 C to help maintain the integrity of our samples until they can be processed Please note that the instrument can maintain samples at lower temperatures if desired e g 4 C but colder temperatures require considerably more power to maintain and are unnecessary in most circumstances in the opinion of MJ Research s
138. tus lights 4 4 cursor buttons 4 3 5 2 display screen 4 3 5 2 layout 2 2 numeric keypad 4 3 5 3 touch pad 4 3 5 3 touch pad buttons 4 3 5 3 using 4 3 Conventions programming 5 4 Copying programs 6 9 Cursor buttons See Control panel Cycler log 7 7 D Deleting a folder 6 1 1 6 12 Deleting a step advanced program 6 6 Deleting a step graphical program 5 20 6 5 Deleting programs 6 10 Display screen adjusting the contrast of 4 3 location of 2 2 Documentation conventions iv In 2 Editing a program 6 4 6 8 advanced 6 6 6 8 deleting a step 6 6 editing a step 6 7 6 8 inserting a step 6 7 selecting steps 6 6 graphical 6 5 6 6 deleting a step 5 20 editing a step 5 20 6 5 inserting a step 5 20 6 5 selecting steps 5 20 Editing a step advanced program 6 7 6 8 Editing a step graphical program 5 20 6 5 Electromagnetic interference A 2 Environmental requirements 3 3 3 5 Error log 7 7 10 2 Error messages 10 3 Ethernet port location of 2 3 Example program 5 8 Extend time option 5 5 5 35 5 36 F FCC warning A 2 File save as window using the 5 17 5 19 File transfer 8 8 Dyad cycler to Dyad cycler 8 8 File utilities 6 8 copying programs 6 9 deleting folders 6 11 deleting programs 6 10 moving programs 6 1 saving edited programs 6 8 Folder creating new 5 18 deleting 6 11 6 12 naming 5 18 Forever incubation advanced program 5 39 graphical program 5 26 Front panel 5 2 See also Control panel Fuses changing 9 3
139. tware that you wish to download e Select lt Update gt e A confirmation screen will appear asking you to confirm your desire to update the software Select lt Yes gt to proceed with the update e A confirmation screen will advise that the update has been completed If any errors occurred during the update select lt Log gt to view the error log If the update was successfully completed restart the Dyad cycler e Select lt About gt from the lt Utilities gt menu to view the currently loaded software version and confirm that the software update has been successfully completed Using the Utilities Mi lt Ping gt This utility can be used to verify that the ethernet software and connection are properly functioning Select lt Ping gt from the lt Utilities gt menu In the Ping window begin by selecting lt Host Names gt and entering the IP address of the computer that you wish to ping All host names must be one word i e no spaces Then select lt Ping Setup gt from the Ping window Ping setup x JONDOEIP 11 11 1111 00 Select a host name using the up down cursor keys Enter the number of times that you wish to ping the host the interval between pings ms and the lost interval the amount of time sec before the request times out Select lt OK gt to return to the Ping window Select lt PING gt to run the ping program The results will be displayed in the DL Sockets TCP IP Kernel window A tally at t
140. u can also directly access this window by selecting lt Network Config gt from the lt Utilities gt menu FIP Server Configuration E M Static M DHCP IP Address GateWay DHS Server OF Cancel M DNA Engine Dyad Operations Manual 8 6 If you have a static internet connection i e the IP address does not change select the Static option Enter your IP Address Net Mask GateWay and DNS Server information using the numeric keypad If you lack this information please contact your network administrator If you have a dynamic internet connection i e a different IP address is assigned each time you access the internet select the DHCP Dynamic Host Configuration Protocol option The IP Address and the other required fields should populate automatically If you are unsure of the type of internet connection available to you please contact your network administrator Once you have finished configuring your network connection select lt OK gt A pop up win dow will appear directing you to power cycler the instrument in order to implement the connection information To update the engine or front end software e Select lt Update Soft gt from the lt Utilities gt menu to access the HTTP Update Software Utility window e Select lt Display Versions gt to view the available software upgrades e Click on the arrow button to the right of the Select Software Version field to display the available versions e Select the sof
141. u will be returned to the File Save As window At this point selecting lt OK gt will write your program to the hard drive Selecting lt Cancel gt will bring up the programming window without saving your program e Select lt OK gt You have returned to the graphical programming window The new program name GRAPH 1 now appears in the Program field The Graphical Programming Window Before we begin entering program steps let s explore the graphical programming win dow The graphical programming window displays the steps of graphical programs in an arrangement of six wide columns separated by seven narrow columns The narrow columns depict the transition phases between steps while the wide columns depict the temperature and or gradient steps included in the protocol A single black line in a wide column indicates a temperature step two black lines represent a gradient step A goto step is indicated by a blue arrow extending from the end of the last step to be included in the loop to the beginning of the first step in the loop The incubation temperature of the step is indicated above the step line and the duration of the step is indicated below the step line i T t f Gradient step emperature step Program amp RAFHH1 _J Lid Mode constant amp 100 00 Step Add Step after Control Mode cac Temp Grad Lolo Forever Goto step Save Save As Save Aun Cancel 5 19 M DNA Engine Dyad Operations Manual 5 20 Se
142. uence the Status window will be visible Block selection menu Programs Command Tools View Utilities Block status line Block is Inactive Block 1 96 User Hame GUEST g Program display box Temperature Time Block 746 C Sample 248 C Lid 23 4 C Cycle 0 Run Instant SIP Pause GRIP Graphs Step 00 00 00 Remaining 00 00 00 Block sample and lid temperatures are displayed for the convenience of the operator along with the current cycle number The time remaining in the current step and in the program are also indicated At the screen bottom the program control buttons lt Run gt lt Instant gt lt Stop gt lt Pause gt lt Skip gt and lt Graphs gt allow the operator global or line by line control of the program currently loaded into memory These will be covered in more detail in Chapter 7 The lt Graphs gt button can be used to display a window that simultaneously and graphically shows sample block and lid temperatures for both Alpha units Note In the Graphs window in the same position there is a lt Status gt button By leaving your cursor in the same position in the window and tapping the touch pad you can toggle between the Status and Graphs windows rapidly The Block Selection menu and Block Status line give information about the block currently selected and its run status The User Name line indicates if a particular user has been se lected
143. ween incubation steps can often help reduce overall reaction times by 10 to 30 and may help reduce production of non specific products The Alpha units use multiple zones of thermal control which allow rapid ramp rates to be balanced with temperature uniformity The Dyad cycler is capable of ramping temperatures in a range of 5 0 C to 105 0 C but no more than 30 C below ambient temperature The ramp rate can be as low as 0 1 C sec or as fast as 3 0 C sec Slower ramp times may be achieved using a combination of incre ment and goto steps Contact MJR Technical Support at 888 652 9253 for details If a ramp rate is not programmed the default will be at maximum M DNA Engine Dyad Operations Manual 5 12 Choosing a Temperature Hold Time Because of the calculated melting temperature Tm of a DNA hybrid DNA polymerase processivity and reaction kinetics it may be possible to generalize conditions regarding thermal cycling protocols However decisions on denaturation annealing or extension hold times will be reaction specific and should be optimized A target temperature can be held for as little as 1 second or up to forever should a protocol require an extended incubation period In graphical programs the maximum programmable hold time for a step is 1 hour 39 minutes and 59 seconds 99 59 with the exception of a forever incubation In advanced programs the maximum programmable hold time is 18 hours with the exception of a f
144. wells Using Oil to Thermally Couple Sample Vessels to the Block With two exceptions see below MJ Research does not recommend using oil to thermally couple sample vessels to the block for the following reasons e Calculated control protocols do not run accurately when oil is used e Oil traps dirt which interferes with thermal contact between vessels and the block Caution If you use oil in the block use only mineral oil Never use silicone oil It can damage the Alpha unit One exception to this recommendation involves the use of volatile radioactive 35S nucle otides A small amount of oil in the block can help prevent escape of these compounds See Appendix 4 B of this chapter for important information regarding safe use of these com pounds in polypropylene tubes and polypropylene and polycarbonate microplates A sec ond exception involves the use of thick wall O 5ml tubes Certain brands of these tubes fit poorly in the block in which case oil may somewhat improve thermal contact Whenever possible use high quality thin wall tubes intended for thermal cycling see Appendix 4 A of this chapter for a tube and plate selection chart Operation W Using the Optional Probe The probe consists of a precision thermistor mounted in a thin walled plastic tube A thin wire encased in a small plastic tube runs from the thermistor to the probe s plug which is inserted into a slot at the back of the Alpha unit see figure 4 3

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