Home

DNA Engine Tetrad® 2 - American Instrument Exchange, Inc.

1. Front Panel Setup 0ccocoocccc seen aannaaien aaa 5 3 Display SCORE RA a e a e e E Y 5 3 Navigation KEYS cido smoren ba e ii a d rw 5 3 Numeric Keypad oo 5 4 Programming Conventions essen hh hh hh hh hh hn 5 4 The Elements of a Program 20000 c cece eee enn n n n n n nn nnn 5 4 Types of Programs 4 22 4619 9 xx euo rox kw Rok md x HR DECR TR CR RR CR TR CREDO TR SR ON DR nn 5 6 Graphical Programs i i du E ura ha x P Roa cac AREA C RU reda 5 6 Advanced Programs iia piede 2a boa bo di O Pda cod bald 5 7 Designing a New Program sss hh hh hh hh hn 5 7 Let s Start with an Example 2usssussmsceksac mk RES WR aa s 5 8 ihe Goto ODOM ento e eee Ace a Ere eR Re s 5 8 Considerations During Program Creation 0 00 cece eee 5 9 Choosing a Temperature Control Mode iullllssssllses 5 9 Calculated Control seese rinane mres aieeaa ena oa a eaa eens 5 9 Er iu ug cosa c o aU CR RR EEE QC E RC n uU EE 5 10 Modifying Block Control Programs for Calculated Control 5 10 Modifying a Program Designed for a Different Machine 5 10 Choosing a Lid Control Mode o 44 520 Sehr arborea 5 11 Choosing a Temperature Ramping Rate 000 eee eee 5 11 Choosing a Temperature Hold Time 000 cee eee eee 5 11 Choosing a Thermal Gradient 000 ccc eee 5 12 Beyond the Example Protocol Other Considerations 5 13 HM DNA Engin
2. Tip To prevent problems with overheating institute a regular program of checking for dust buildup particularly for robotics installations Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Maintenance W 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 methods monitoring and disposing of contaminated materials Changing the Fuses The circuits in the DNA Engine Tetrad 2 cycler are protected by four fuses 6 3A fast acting 5 x 20 mm When a fuse blows the cycler immediately shuts down and cannot be turned back on The machine records the event as a power loss If a pro tocol is running when a fuse blows the machine will resume the run when the fuse is replaced and the power restored which means that live power may still be available inside the unit even when a fuse has blown or been removed Never open the DNA Engine Tetrad 2 base You could receive a serious electrical shock Opening the base will also void your warranty f Warning The DNA Engine Tetrad 2 cycler incorporates neutral fusing 1 Disconnect the power cord from the back of the instrument Move the power switch to the 0 off position 2 Insert a small flat head screwdriver into the slot in the center of the fuse plug figure 9 14 and gently turn Th
3. 5 72 30 6 GOTO 3 39X 7 72 7 00 8 4 9 END 2 step nested primer protocol NEST PR2 1 95 4 00 2 95 30 3 65 30 4 GOTO 2 39X 5 72 7 00 6 4 7 95 30 8 65 30 9 GOTO 7 39X 10 72 7 00 11 4 12 END 3 step nested primer protocol NEST PR3 Step Temp Time 1 95 4 00 2 95 30 3 55 30 4 72 30 5 GOTO 2 39X 6 72 7 00 7 4 8 95 30 9 55 30 10 72 30 11 GOTO 8 39X 12 72 7 00 13 4 14 END 2 step long PCR protocol using iTaq polymerase LONG 2 1 95 4 00 2 95 30 3 65 3 00 Ext 15s cycle 4 GOTO 2 34X 5 72 7 00 6 4 7 END 3 step long PCR protocol using iTaq polymerase LONG 3 1 95 4 00 2 95 30 3 55 30 4 72 3 00 Ext 15s cycle 5 GOTO 2 34X 6 72 7 00 7 4 8 END C 2 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Index A About utility 8 2 Accessories See Moto Alpha Unit Accuracy See Thermal accuracy Adding a step advanced program 6 7 graphical program 5 21 Advanced mode programming 5 27 5 37 Advanced program deleting a step 6 6 editing 6 6 6 8 See also Editing a program extend time option 5 33 goto step entering 5 34 gradient step entering 5 31 5 32 increment temp option 5 36 5 37 inserting a step 6 7 lid control step entering 5 35 opening 6 2 6 3 slow ramp option 5 35 specifications 5 7 temperature step entering 5 28 5 31 Advanced programming window 5 28 Air intake vents location of 2 2 2 3 Air supply requirements ensuring adequate air s
4. DNA Engine Tetrad 2 Peltier Thermal Cycler Operations Manual PTC 0240 DNA Engine Tetrad 2 Cycler DNA Engine Tetrad 2 Peltier Thermal Cycler PTC 0240 DNA Engine Tetrad 2 Cycler Operations manual Supports software version 4 0 Copyright 2005 Bio Rad Laboratories Incorporated All rights reserved Reproduction in any form either print or electronic is prohibited without written permission of Bio Rad Laboratories Inc Alpha Chill out CleanBox Concord Disciple Desktop DNA Engine DNA Engine Dyad DNA Engine Tetrad Dyad Disciple Hard Shell Hot Bonnet iProof iTaq Microseal Moto Alpha Multiplate Power Bonnet Slide Chambers and Tetrad are trademarks belonging to Bio Rad Laboratories Inc Teflon is a trademark of E I DuPont de Nemours and Company Windows is a trademark of Microsoft Corporation NOTICE TO PURCHASER This base unit Serial No in combination with its immediately attached Bio Rad sample block module s constitutes a thermal cycler whose purchase con veys a limited non transferable immunity from suit for the purchaser s own internal research and development and for use in applied fields other than Human In Vitro Diagnostics under one or more of U S Patents Nos 5 656 493 5 333 675 5 475 610 claims 1 44 158 160 163 and 167 only and 6 703 236 claims 1 7 only or corre sponding claims in their non U S counterparts owned by Applera Corporation No right is conveyed expressly by
5. 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 example and Time enter 30 seconds as an example e Select Increment Temp e Select Set Parameters for the Increment Temp option The Increment Temperature window appears Increment Temperature EZ M Increase Decrease by C per cycle OK Cancel e Select Decrease e Position the cursor over the by C per cycle field and select Enter 0 2 in the field e Select lt OK gt You would expect the program step as included in a protocol to appear as follows 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 5 36 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W The final program shou
6. een a Rosie i QUE E oe it NE O X s e n y K de 1 Mi s SY 4 x s dA i M ee c ts i i date of issue ni ex uy m i i to which this declaration relates is in conformity to the following standards Bio Rad Laboratories Inc 1000 Alfred Nobel Drive Hercules California or normative documents 94547 U S A declares that the product following the provisions of the 73 23 EEC 89 336 EEC amp 93 68 EEC Directive This product is imported into the EU by Bio Rad Laboratories Ltd Bio Rad House Maxted Road Hemel Hempstead London area Hertfordshire i 3 PTC 0240 The DNA Engine Tetrad 2 Thermal Cycler HP2 7DX England EN61326 CLASS A 13 September 2004 IEC61010 1 1 H x t i ey a E h e T x i 4 M K K r VO Buy ae SCR i ma j fie Oy ti ci i d A a s 26 4 d At he zn d s i d eS zh e e ex S e Sel E Ed um i x o MM AN MANC M O jM ni EN TA EN UM i A a ea UA 41 e 4 BR A BR 4 C S S tete EEN aestu EM Steet Cu ooo NOE eee BRS AA UA UN UA A UR S SDN NU SAN QUU VAR QA SAN NORRIS RB VAR ATA SR Ql MAN NL VAN A AUI AN MEAN SA RN NA VAN UL VA NL AN NU VAR NI ON NL VAN UR VAR ATA RN ATA VANS E OR RA SAN RB VAR OR MAN ME ECO oo Kol Ee heh AN peer eia td o MEIE SIE Ho atero telat E td neonato peu ta Fool SS E i Ae tole SER tf a
7. Cleaning the DNA Engine Tetrad 2 Cycler ooooooocoocoo 9 2 Cleaning the Chassis and BiGCK xoa cc Re c ee Dewees 9 2 Cleaning the Air Ventss sxsw vae ck Fe Rees qnae a id 9 2 Cleaning Radioactive or Biohazardous Materials Out of the Block 9 3 Changing the Fuses 0 ooccoococ een hh hh a nnn 9 3 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual 9 2 Cleaning the DNA Engine Tetrad 2 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 reason ably clean contaminant free environment is recommended To clean block wells use swabs moistened with water 9596 ethanol or a 1 100 dilution of bleach in water see the Slide Chambers Alpha Unit Operations Manual for instructions on cleaning the Slide Chambers slide slots If using bleach swab wells with water after ward 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 conce
8. Error log 7 7 10 2 Error messages 10 3 Example program 5 8 Extend time option 5 5 5 36 F Index M Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com In 3 DNA Engine Tetrad 2 Thermal Cycler Operations Manual File save as window using the 5 17 5 19 File transfer 8 8 From one cycler to another 8 8 File utilities 6 8 copying programs 6 9 deleting folders 6 11 deleting programs 6 10 moving programs 6 11 saving edited programs 6 8 Folder creating new 5 18 deleting 6 11 naming 5 18 Forever incubation advanced program 5 37 graphical program 5 26 Front panel 5 2 See also Control panel Fuses changing 9 3 G Goto step 5 5 5 8 advanced program 5 34 5 35 graphical program 5 25 Gradient step 5 5 advanced program 5 31 5 32 graphical program 5 24 5 25 Gradient using to optimize 5 12 Gradients gradient preview window 5 32 specifications 2 5 accuracy 2 5 calculator accuracy 2 5 In 4 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Index M column uniformity 2 5 lowest highest temperature 2 5 temperature differential range 2 5 Graphical mode programming 5 15 5 26 Graphical program adding a step 5 21 deleting a step 5 21 editing a step 5 20 6 5 forever incubation entering 5 26 goto step entering 5 25 gradient step entering 5 24 opening 6 4 selecting a step 5 20 specifications 5 6 5 7 temperature step entering 5 21 5 24 Graphs window 5 14 H Hold time 5 11 Hot Bonnet lid 4 8
9. Go to step number Additional Number of Cycles OK 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 The program 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 3 Gradient from 45 C to 65 C for 00 03 00 4 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 Moto Alpha motorized lid 5 34 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs 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 the Moto Alpha unit motori
10. 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 conse quence of the geometry of the Peltier Joule heaters that underlie the block and is normal Rest assured that the temperatures displayed are quite accu rate for each well in that column 0 4 C of actual column temperature e Select lt Close gt e Select OK The program listing should now appear as follows Since the program step listing field is limited please use the directional arrow keys to navigate 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 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 5 32 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W 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 enz
11. USB B port for PNOSTSTSTSTSTSTSISFSESI ISI S181 S767 62 5757 57 67 S75 USB A port for mouse SIS 8782825282782 826787578782 828 JUSINGISISISISISISISISISISISIS OOOO ATA ATA TA ATA TONOS SISISISISISQISISISISISISISISISISI 26 6799576767 67 6 6 5267 67 6 64 852662525757 6 6767 BOS S2 S7 S S2 S2 S2 S7 S2 VAESISTSUSIS 000000000 00000000 00000000 00000000000000000009 o o 600000000000000000 oooooooQ 00000000 oo00000000 99000000 000000000 909000000 oo0000000 0000000 000000000 DNA Engine Tetrad 2 cycler rear oooo0o00000006 oooooooooooooooooo Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com DNA Engine Tetrad 2 Cycler Bottom View Figure 2 4 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual Compatible Alpha Units Single Block Models 60 Single 96 Single 384 Single Flat Block Dual Block Models 30 30 Dual 30 48 Dual 48 48 Dual Slide Block Slide Chambers Alpha unit Moto Alpha Unit Holds 60 x 0 5 ml tubes Holds 96 x 0 2 ml tubes or one 96 well microplate Holds one 384 well microplate Holds customer designed adapter through four screw down points Holds 2 x 30 x 0 5 ml tubes Holds 1 x 30 x 0 5 ml tubes and 1 x 48 x 0 2 ml tubes Holds 2 x 48 x 0 2 ml tubes or half plates Holds 2 x 16 standard slide
12. 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 using 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 Utilities Command View Block 14 30 Block status line Incubating at 37 0 C User Name GUEST r Temperature Time Block 369 C Step 00 00 08 Sample Remaining Forever Lid 46 3 C Cycle 1 Skip 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 continue Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 7 3 ll DNA Engine Tetrad 2 Thermal Cycler 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 ru
13. modes However after selecting OK to accept any changes and exit the Mode Selection window you will return to the advanced programming window Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 27 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Program Untitled Lid Control Mode Tracking at 5 C above End H E Step After Temp Gradient GoTo 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 pro gramming session 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 e Select Save As 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 window The following steps will appear to indicate your progress with the program ADV 1 Temperature Control Mode Calculated Lid Control Mode Const
14. 4 9 adjusting lid pressure 4 9 l Increment temp option 5 5 5 36 5 37 Inserting a step advanced program 6 7 Inserting a step graphical program 5 21 6 5 Instant incubation 7 2 K Keyboard virtual 5 18 Keypad 5 4 L Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com In 5 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Layout control panel 2 2 Lid control methods constant mode 5 11 off 5 11 tracking mode 5 11 Lid control step advanced program 5 5 5 35 Loading sample vessels 4 10 Maintenance 9 2 Microplates removing 3 6 selecting 4 7 Microplate sealing mat 4 9 4 12 Microseal A film 4 8 4 12 Microseal B adhesive seals 4 9 4 12 Microseal F foil 4 9 4 12 Microseal P pads 4 9 4 12 Microseal P pads 4 9 4 12 Mode selection window 5 16 5 27 Monitoring the run 7 6 Moto Alpha unit 2 4 5 38 Mouse device connecting 3 3 Moving programs 6 11 Multiple programs running 7 9 N Network config utility 8 6 8 7 Numeric keypad See also Control panel location 2 2 O In 6 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Opening a program 6 2 6 4 advanced mode 6 2 6 3 basic mode 6 4 Operation turning the Tetrad 2 cycler on 4 2 P Packing checklist 3 2 Password utility 8 5 Pausing a run 7 8 Ports 2 5 4 4 RS 232 8 3 Power cord plugging in 3 2 Power supply requirements 3 4 acceptable power cords 3 4 Program adjusting for sample vessel type 4 6 4 7 advanced 5 27 5 37
15. 5 17 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual e Select New Folder gt els s x s 0 1 E a EL GM 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 letters and numbers should be entered e Using the external mouse select the characters F O L D E R 1 in suc cession The backspace key can be used to correct any mistakes e Select OK 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 You 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 5 18 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W Folder Passw
16. A GE OR TA LT A ELE Ti A CEDE V ELE REIR V CERA a ad EN A CER EI AE V EET 4 4 e E 4 E 4 z 4 AI E P E 4 4 Fee tone VUA EE Ml otto Mir Merit Mco Meco Moron Ml arneses GEOCOT HES rape lociones ELLA AREIS Soo Moor RACES ELE E ERIS Se aternaas GIA EE ERIS MILAN tag EO RS IS E ESTER a a TE pe ah hi A OCLC ICC NR QR C NN NE E QC OC QOO ORE dn pcc NER EE ER EM E E e E B ee e E e Ee Ee E E M CE eee c t SN a Nox RUM MESES QN SOUS EE zz MAT TA n ziii th RUN SCR u NA E ON i Hi 11147 revB E qa MM oj l i Sy j A y 1 J 7 t E B RT i S A V j h i a o x Mi n i i AN 4 Vice President jh 1 i ui i i l nas Brad Crutchfield mt a T UE y i Ny i ri iit l Ta i Ux QS a AE Ms a M 1 i NG KOY NS AAA Nd Hs DENT OK e i z a i amp Y i d Y y ES F a i s P i j CX i 7 AN e JM N A al a ns i E E 3 ONOS A 2 AKAN Mi Pe m AY un X Men i amp Y i j wk ES a AO AAA t bin N RRA a Bi SAMO E Sa XX 25 NON de NUES NN o We Hit uy t i E i itt NORO ie id AU 1G j 3 c eae X T Hs Jn i gt q i O um c O O EE Ya O c O E U L o O e RE n n xe j Mi I MN Oy sd Hl y i j TN 3 y
17. A SO 2 vox ya tes Lilo pe die 6 7 Editing a SIep sionista aa 6 7 File Utilities 2222225559 S24 5e tta LE ned eee adiu e gu ered quid atop au dat 6 8 Saving an Edited Programi sssi erasia a E ea 6 8 Copying a Program 23 2 sasarap b dd er de did ide el xad 6 9 Deleting a Program se creadas ribera va 6 10 Moving a Progra 32e arar A s de sob 6 11 Deleting a FOU BE v oos od dr acu A der aider a 6 11 E DNA Engine Tetrad 2 Thermal Cycler 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 Tetrad 2 programs The programming conventions listed in Chapter 5 will also be used here Please review these before proceeding Opening a Program Several useful protocols covering a variety of applications are preprogrammed into the DNA Engine Tetrad 2 cycler and stored in the MAIN folder These programs are listed in Appendix C Any of the protocols saved on the DNA Engine Tetrad 2 cycler whether created by the user or preprogrammed in the factory can be opened for editing or running From the Status window menu bar e Select Programs Select Open An additional menu appears allowing you to choose Advanced Mode or Basic Mode Opening a Program in Advanced Mode To open our example advanced program ADV 1 e Sele
18. Air flowing into intake vents is not Check temperature of air entering Gili Ss air intake vents following proce dure on p 3 4 If higher than 31 C use Table 3 1 to troubleshoot and remove cause s Dust and debris clogging Failure to regularly check for Remove light collections with up air intake vents buildup damp cloth Vacuum out heavy collections Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 13 Appendix A Warranties The DNA Engine Tetrad 2 PTC 240 thermal cycler is warranted against defects in materials and workmanship For specific Warranty information contact your local Bio Rad office If any defects should occur during the warranty period Bio Rad will replace the defective parts without charge However the following defects are specifically excluded 1 Defects caused by improper operation or by improper packaging of returned goods 2 Repair or modifications done by anyone other than Bio Rad Laboratories 3 Use with tubes plates or sealing materials not specified by Bio Rad Laboratories for use with the DNA Engine Tetrad 2 thermal cycler 4 Deliberate or accidental misuse 5 Damage caused by disaster 6 Damage due to use of improper solvent or sample The warranty does not apply to fuses For inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument For technical service call your local Bio Rad office or in
19. Alpha unit only Gradient accuracy 0 3 C of programmed target at end columns 30 seconds after the timer starts for the gradient step NIST traceable Column uniformity 0 4 C well to well within column within 30 seconds of reaching target temperature Calculator accuracy 0 4 C of actual well temperature NIST traceable Lowest temperature 30 C for gradient Highest temperature 105 C for gradient Temperature differential 1 24 C range Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 2 5 3 Installation Unpacking and Moving the DNA Engine Tetrad 2 Cycler 3 2 Packing Checklist si cid ecw id dd a PESE E eee 3 2 Setting Up the DNA Engine Tetrad 2 Cycler 00 cece cece ee eee eee eens 3 2 External Mouse Device 00 ccc cece ees 3 3 Environmental Requirements 00000 cece ee ee ees 3 3 Power Supply Requirements 000cc cece ee eee eee 3 4 Air Supply Requirements 00 0c ee 3 4 Ensuring an Adequate Air Supply cines cede aea A wee ARA 3 4 Ensuring That Air is Cool ENOUGH e eserse dos a a ai seas axe 3 5 Requirements for Robotics Installations eel 3 5 384 Well Microplate Specifics oooocooooocna es 3 6 3 2 DNA Engine Tetrad 2 Thermal Cycler Operations Manual Unpacking and Moving the DNA Engine Tetrad 2 Cycler Please read the Unpacking Instructions for the DNA Engine Tetrad 2 Thermal Cycl
20. Bio Rad Microseal Microseal Microseal Microplate Microseal Microseal Strip Chill out 0 2ml 0 5ml Catalog A film B seal F foil sealing mat P pad P pad caps wax MSA 5001 MSB 1001 MSF 1001 223 9442 MSP 1001 MSP 1002 TCS serie CHO series Hard Shell skirted HSP ries 384 well microplates Se Microseal skirted 384 well microplates niue skirted HSP series 96 well microplates Microseal skirted MSP series 96 well microplates Multiplate unskirted MLP series 96 well microplates MLL series Multiplate unskirted um 48 well microplates Multiplate unskirted 25 well microplates MLP 2501 Multiplate unskirted JEN ey well microplates PEU E v Concord Edme CON 9601 v v v 96 well microplates 0 2ml strip tubes TBS series 8 strip amp 12 strip TLS series 0 2ml individual v v 0 5ml individual 3 Concord microplates are made from polycarbonate plastic which is prone to poor sealing and vapor leakage during stringent thermal cycling MSP series Since plates may adhere tightly to the Microseal P pad this seal is only recommended for use with the Moto Alpha motorized unit which has the ability to hold the plate in the block 4 12 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Operation M Appendix 4 B Safety Warning Regarding Use of S Nucleotides Some researchers have experienced a problem with radioactive contamination when using S in thermal cycler
21. Config These utilities allow you to update both the software controlling the DNA Engine Tetrad 2 cycler s hardware i e engine software and the user interface programming software i e front end software 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 DNA Engine Tetrad 2 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 Update Soft and Network Config utilities To view the available software upgrades and or perform an upgrade begin by estab lishing internet access via an Ethernet 10BaseT connection Connect the ethernet cable to the ethernet port located at the rear of the DNA Engine Tetrad 2 cycler see figure 2 3 Then select Update Soft from the Utilities menu The HTTP Update Software Utility window appears HTTP Software Update Utility select Software Version Display Versions Front End Version 4 00 Sept 30 2005 Dyad 220 Engine Code Version 3 3G Time Dut Update Llog NetworkConfig By selecting lt Network Config gt you may view your current information By default DHCP Dynamic Host Configuration Protocol is enabled Once you have reviewed your network connection select lt Exit gt To update the eng
22. Tetrad 2 cycler to power four bays Clear will deselect all bays Select lt OK gt to exit or lt Cancel gt to disregard any changes made in this panel Bay Configuration x r Select Bays Bay 2 Bay 3 Bay 4 Select All Clear OK Cancel lt Bay Identification gt This utility allows the user to identify particular bays Each cycling bay in the DNA Engine Tetrad 2 cycler has a blue LED located next to it Select a particular bay and specify the Number of Times to Flash LED This utility is useful trying to identify which of the cycling bays is considered Bay 3 or Bay 4 when running programs Comm Config For DNA Engine Tetrad 2 cycler users this utility is grayed out and unavailable It is available only to Disciple Desktop owners who run the Dyad Disciple instrument using the same software Service Many of the utilities within the Service submenu are intended for use by Bio Rad personnel only Password allows the field service technicians and distributors to enable all service mode utilities Block Log allows the user to view the last recorded quadrant error to be relayed to Customer Service Folder Password becomes enabled when a service password is entered Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 8 5 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual lt Network gt Update Soft amp Network
23. 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 incuba tion temperature of the step is indicated above the step line and the duration of the step is indicated below the step line Gradient step Temperature step Program GRAPH 7 Lid Mod constante 100 00 Step 1 Add Step after Control Mode carc Temp Grad Golio Forever Save Save As Save xHun Cancel Goto step Selecting a Step When a step is selected the line s or arrow depicting that step will turn red Select a step by positioning the cursor in the step s column and 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 in the desired field and click once to display an insertion point or click twice to highlight the entire field 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 Click the mouse once to accept the change and move the cursor 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
24. 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 10 oz 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 4 Spray for about 1 second onto the block Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com A Operation Turning the DNA Engine Tetrad 2 Cycler On o oooccocooc eee 4 2 Using the Control Panel 4s xacin O3 a x REX ads 4 3 Display Screen us aa eoo asas ls a ra 4 3 Operation NOUS 2 usus add reer ers edo dur dpa eda i sehen ddl ddr WR 4 3 Blpck Status LIIS a iue aa e Den dar oa da a Rice Qo ddr reat a ae dora Re 4 4 Using the Data Ports rl I RIED I x EE ER 4 4 Operating Alp
25. copying of the SOFTWARE PRODUCT by LICENSEE and shall terminate upon LICENSEE s discontinued use of the SOFTWARE PRODUCT or the transfer of the SOFTWARE PRODUCT as provided in Paragraph 2 1 d whichever occurs earlier provided however that this AGREEMENT and the license granted hereunder shall survive and continue as to the transferee 6 2 Termination This AGREEMENT and the license granted hereunder may be terminated by BIO without prejudice to any other rights by written notice to LICENSEE only in the event that LICENSEE is in breach of any material provision of this AGREEMENT which breach is not cured after reasonable notice In the event of termination by BIO LICENSEE shall destroy all copies of the SOFTWARE PRODUCT in LICENSEE s pos session and all of its component parts LICENSEE may terminate any license granted hereunder by returning the SOFTWARE PRODUCT and any documentation including the MANUAL to BIO 7 GENERAL PROVISIONS 7 1 Entire Agreement This AGREEMENT is the entire agreement and understanding of the parties hereto with respect to the SOFTWARE PRODUCT and supersedes all prior oral written or other representations and agreements This AGREEMENT may only be amended in writing by an authorized agent of BIO 7 2 f any provision of this AGREEMENT is held invalid the offending clause will be modified so as to be enforceable and as modified shall be fully enforced and the remainder of the AGREEMENT will continue in f
26. 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 Cycling and even automatic shut downs Special attention should be paid to airflow and air temperature in robotics installations of DNA Engine Tetrad 2 cyclers Ensuring an Adequate Air Supply e Do not block the air intake vents Position the DNA Engine Tetrad 2 cycler at least 10 cm 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 e 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 completely clogging up Check for dust and debris every few months and clean the intake vents as needed Remove light collections of dust with a soft bristle 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 e Use a solid non perforated support material when using the DNA Engine Tetrad 2 cycler on a wire rack Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Installation Mi Cause Possible Remedies Air circulation is poor
27. extend time option 5 33 goto step entering 5 34 gradient step entering 5 31 5 32 increment temp option 5 36 5 37 lid control step entering 5 35 slow ramp option 5 35 temperature step entering 5 28 5 31 advanced options beep on target 5 6 extend time 5 5 increment temp 5 5 slow ramp 5 6 copying 6 9 Index M Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com In 7 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual deleting 6 10 designing choosing temperature control method 5 9 choosing lid control mode 5 11 choosing temperature gradient 5 12 See also Gradients choosing temperature hold time 5 11 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 5 temperature step 5 5 graphical 5 15 5 26 adding a step 5 21 deleting a step 5 21 editing a step 5 20 6 5 forever incubation entering 5 26 goto step entering 5 25 gradient step entering 5 24 graphical programming window 5 15 selecting a step 5 20 temperature step entering 5 21 5 24 inaccessible features 7 8 monitoring the run In 8 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 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 multi
28. fa th ANLE IS eoa tet Nee ti Fol Ay refi teh SS A A o o A Pea th a ed a ose AD C So Mice aou c eie Duet deme rx eden detener depen o e Pv ede c erii dete emend cii equidem derum Piet qui attende iei o Petr dete e tates EN NS NS A EE SS NES NES NS NS NES NES eae NS TAN NES NR eee NES NES TA reo coe ar reo rec reo rhe rec reo XA NR ES Technical Service Call your local Bio Rad office or in the U S call 1 800 4BIORAD 1 800 424 6723 Life Science Group Bulletin 05184 US EG Rev E Bio Rad Laboratories Inc Web site www bio rad com USA 800 4BIORAD Australia 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 2527 3454 Canada 905 712 2771 China 86 21 6426 0808 Czech Republic 420 2 41 43 05 32 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 2398112 3 4 5018111 6450092 93 Israel 03 951 4127 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Latin America 305 894 5950 Mexico 55 52 00 05 20 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 095 721 1404 Singapore 65 64153188 South Africa 00 27 11 4428508 Spain 34 91 590 52 00 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 2578 7241 United Kingdom 020 8328 2000 05 0868 1005 Sig 1204
29. 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 e 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 e Change the time from 30 to 25 e Select lt OK gt Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 6 7 E DNA Engine Tetrad 2 Thermal Cycler 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 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 dif ferent filename In Chapte
30. inserting or Caution Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2 removing an Alpha unit or operating the DNA Engine Tetrad 2 cycler The Alpha units must be installed prior to powering up the DNA Engine Tetrad 2 cycler see the Operating Alpha Units section below for installation instructions The power switch is located at 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 1 minute 30 sec onds During the boot sequence the user is presented with several options including 1 Selftest Choose number 1 on the keypad to instruct the DNA Engine Tetrad 2 cycler to perform a diagnostic system test and report any errors 2 Send Files Choose number 2 on the keypad to prepare the cycler to transfer stored program files to another DNA Engine Tetrad 2 cycler see Chapter 8 for instructions on transferring program files 3 Receive Files Choose number 3 on the keypad to prepare the cycler to receive stored program files from another DNA Engine Tetrad 2 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 boot up the DNA Engine Dyad Tetrad 2 logo screen is briefly displayed The DNA Engine Tetrad 2 cycler s status window will then be vi
31. lid surface at a specific temper ature 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 This mode incorproates a temperature offset of the heated inner lid by a minimum specified number of degrees Celsius in comparison to the temperature of the sample block Tracking 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 between incubation steps can often help reduce overall reaction times by 1096 to 3096 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 DNA Engine Tetrad 2 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
32. mouse In some instances selection is also possible by clicking the lt lt Enter gt gt button This instrument system is labeled For Research Use Only 1 Introduction Meet the DNA Engine Tetrad 2 Cycler ooooooooooonorran eese Using This Manual ee ER REV A eee ees Important Safety Information cococcoccncn err B DNA Engine Tetrad 2 Thermal Cycler Operations Manual 1 2 Meet the DNA Engine Tetrad 2 Cycler Thank you for purchasing an DNA Engine Tetrad 2 cycler Designed by a team of molecular biologists and engineers the DNA Engine Tetrad 2 cycler delivers multi block thermal cycling with superior thermal performance The programmable DNA Engine Tetrad 2 cycler with its 4 bay chassis is ideal for running multiple proto cols and accommodating multiple users The DNA Engine Tetrad 2 cycler offers the following features Interchangeable sample blocks the Alpha unit family accommodate a variety of tubes microplates and slides Hot Bonnet heated lid for oil free cycling or the Moto Alpha unit for auto mated systems Intuitive DNA Engine Tetrad 2 system software with user friendly interface for programming editing file management and much more Choice of calculated temperature control for highest speed and accuracy or of block or temperature control for compatibility with protocols designed for a variety of instrument types Instant Incubate feature for continuous temperature incubations
33. protocol change if needed Check screen for probe failure error message Probe may need servicing or replacing Call Bio Rad Laboratories or your local distributor Fill probe tube with correct amount of oil see p 4 12 Test a complete 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 seaing Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 11 HM DNA Engine Tetrad 2 Thermal Cycler Operations Manual Table 10 2 Protocol Difficulty Error Message Cause and Result Reaction is working but Primer dimer material often pro broad low molecular weight band is seen in gels Reaction working but unexpected extra prod ucts or smear is seen duces a broad band in the 100 bp region of gels Nonspecific hybridization occurring 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 reaction product s no need to change anything Minimize primer dimer production by designing primers with no 3 self complementary Reoptimize magnesium concentra tion and annealing temperature to maximize desired product and m
34. the Run Run Status Information in the Temperature Time and Cycle fields of the Status window will indi cate that your program is running To graphically display the run conditions go to the Graphs window 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 esti mated Time Remaining in the program and the amount of Time Elapsed since the program was initiated are also indicated Program ADY 2 Active User Name GUEST aj ey e Time Remaining 01 57 30 Time Elapsed 00 19 56 Ming a Gamma se Ps in Sec M Block Y Sample M Lid Cycle 4 of 30 Users can scroll through the temperature display graph Left and right arrow buttons allow the leading edge dashed vertical green line to be advanced or subtracted as long as the minimal time scale T 9 40s 1min has been sat isfied The magnify buttons with the and symbols allow the user to change the scale at which they view the graph The minimum default is 9 40 Each time you click the the scale doubles 19 20 38 40 77 20 154 40 and 309 20 Inversely will bring you from a larger scale graph toward the minimal scale 9 40 Note If communications restart or power failure occurs the graph will be cleared and start from time 0 This does not mean that the protocols have be
35. the United States call 1 888 424 6723 or visit us on the Web at www bio rad com Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com A 1 Appendix B License Agreement END USER AGREEMENT AND LICENSE FOR BIO RAD DNA ENGINE TETRAD 2 SOFTWARE ALL VERSIONS IMPORTANT PLEASE READ THE FOLLOWING LICENSE AGREEMENT YOU HAVE PURCHASED THE Bio Rad DNA Engine Tetrad 2 Thermal Cycler WHICH INCLUDES CERTAIN EQUIPMENT AND INSTRUMENTATION AS WELL AS A LICENSE TO COMPUTER SOFTWARE This End User License Agreement AGREEMENT is a legal agreement between you either an individual or a single legal entity hereinafter LICENSEE and Bio Rad Laboratories Incorporated Bio for the Bio Rad DNA Engine Tetrad 2 Thermal Cycler the CYCLER which includes certain equipment or instrumentation BIO pro prietary computer software SOFTWARE PRODUCT and may include online or electronic documentation associated media and printed materials including an operator s manual MANUAL The CYCLER also contains software owned and pro duced by third parties THIRD PARTY SOFTWARE which is licensed for use on the equipment and instrumentation of the CYCLER only and the terms of the SOFT WARE PRODUCT license below apply BY OPERATING THE CYCLER OR INSTALLING COPYING OR OTHERWISE USING THE SOFTWARE PRODUCT OR ANY UPDATE THERETO YOU AGREE TO BE BOUND BY THE TERMS OF THIS AGREEMENT AND ANY ACCOMPANYING AGREEMEN
36. the control panel such as lt lt ENTER gt gt lt lt 1 gt gt and lt lt LEFT gt gt e lt gt will be used to indicate windowed menu items or buttons such as PROGRAMS lt RUN gt and lt UTILITIES 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 synonymous with click on point and click and any phrase ology implying selection of menu or option items with the mouse The Elements of a Program Tetrad 2 programs consist of a combination or series of steps and setup parameters that represent protocol requirements Note The procedures involved in actually entering these steps will be described in sub sequent pages but please familiarize yourself with the types of steps used to create Tetrad 2 programs The considerations behind choosing various elements will be explained further in the Considerations During Program Creation section The following is a summary of the individual program elements and their basic functions Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W Temperature Control Mode This parameter defines the temperature control algorithm used during the program run The three different modes include Calculated and Block Due to the expected lag of sample temperature behind block temperature the DNA Eng
37. the first few times through as they will probably save trou bleshooting time later Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 7 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Example Program Let s start with an example Assume you have the necessary components for a 30 cycle amplification reaction and you have calculated the annealing temperature of your oligonu cleotide to be 60 C Please note that we recommend 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 pow ame d e 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 1 2 3 4 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 5 8 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 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
38. time or number of cycles 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 be rejected and the default value or last valid value will reappear Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W 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 selected 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 GoTo 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 instructions on adding specific types of steps and entering step parameters Now let s begin entering the steps for our example program 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 1 92 C for 30
39. 00 424 6723 e www bio rad com Running Programs W The Run Program Window Program MYFIRST um BUEST E r Select Blocks Select All Clear Indicates Block Type Used for Calc Mode DK 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 DNA Engine Tetrad 2 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 EZ Reaction Volume ul Enter a value between 10 pl and 100 pl F Polycarbonate Plates Iv Polypropylene Plates and Tubes OK Cancel e Enter the reaction volume of your samples and select the type of reaction 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 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 7 5 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual During
40. 00 424 6723 e www bio rad com 4 13 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 4 14 The Solution Substitute the low energy beta emitter 3P in cycle sequencing P nucleotides are not subject to the same kind of chemical breakdown as S nucleotides and they have not been associated with volatile breakdown products If 58 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 0 5 ml 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 isotope Consult your radiation safety officer for his or her recommendations If mild cleaning agents do not remove radioactivity harsher cleaners may be used occasionally 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 thoroughly rinsed away within a few minutes of application They can eat away the surface finish of the blocks A Caution Harsh cleaning agents such as those above are corrosive and must be Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs
41. 10 7 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual Problems in Power Up Should a DNA Engine Tetrad 2 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 Turn power on Plug cycler into Is the unit Did you hear 220V AC plugged in a beep Alpha units in the correct position and are handles locked in place Secure Alpha units Does the initialization screen appear Are fuses good Replace fuses 5x20 mm 6 3A Unit defective return for service Was 1 hit to enter Selftest Selftest Does the Communications Splash screen appear Unit ready 10 8 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Troubleshooting Mi Problems with System Performance Should you encounter problems with menu navigation front panel manipulation or any performance aspect of a DNA Engine Tetrad 2 cycler that has successfully pow ered 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 ID Select 1st block Re boot and run self test program Are temperature
42. E mmu 8 6 Gradient Calc aiii REI a 8 7 Transferring Program Files leeren n n n n n nnn 8 7 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual In previous chapters you ve learned how to install and operate the DNA Engine Tetrad 2 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 DNA Engine Tetrad 2 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 Utilities has five options with submenus Control Config Service Network and Grad Calc as well as the About dialog box About When this item is selected a screen such as the following will appear Version 4 00 16 Sept 30 2005 x DNA Engine Dyad and Tetrad 2 Peltier Thermal Cyclers Serial Number Front End Software Yersion 4 00 16 Sept 30 2005 Engine Software Versions Bay 1 3 3G Bay 2 3 36 This screen indicates the DNA Engine Tetrad 2 system software version The software version information on this screen can be used to determine if an update is necessary Select OK or click on the x in the upper right corner to exit from this screen 8 2 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Using the Utilities M
43. 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 protocols and reactions see Current Protocols in Molecular Biology F Ausubel et a eds John Wiley amp Sons 10 10 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Problem Cause Troubleshooting Mi Action No reaction products obtained 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 nat filled with correct amount of oil Reaction mix contains an inhibitor e g heme from blood Reaction vessels not making good thermal contact with sample block Re run reaction using correct protocol Use List utility to check protocol s temperature control method tem peratures and times Check reaction assembly protocol ensuring that mixture contains appropriate components in correct concentrations Use 92 C for denaturation Only rarely are temps higher than 94 C required however Check for appropriate annealing temperatures of primers using available computer programs or empirical testing Use List utility to check tempera ture control method for
44. Gradient GoTo Lid Temp Grad Golo Iv Forever a Delete Step Save Save Run Cancel Delete Step Save Run Save Save As Cancel Basic program Advanced program Graphical Programs Creating a basic graphical program is desirable if you prefer the graphical program ming interface and 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 5 6 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W In addition to the speed with which they can be entered another benefit of graphical programs is that they can be quickly edited This can be particularly useful if you tend to repeatedly run the same general protocol with limited changes e g varying annealing temperatures Advanced Programs Advanced programs offer all of the Tetrad 2 programming features with the excep tion 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 modi
45. PLATE select Save As 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 cannot be run 5 26 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W Entering a Program Using Advanced Mode e Select Programs Drop down submenus appear including Open New Copy Move Delete Delete Folder and New Folder e Select New An additional menu appears allowing you to choose Advanced Mode or Basic Mode e Select Advanced Mode 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 xl Temperature Control Mode v Calculated Block In Sample Probe Lid Control Mode v Constant Tracking Tort Power Lid Parameters 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
46. Provide more space around machine or adjust room ventilation Ambient air temperature is high Adjust air conditioning to lower ambient air temperature Machine is in warm part of room Move machine away from or protect machine from such heat sources as radiators heaters other equipment or bright sunlight Machines are crowded Arrange machines so that warm exhaust air does not enter intake vents Ensuring That Air Is Cool Enough e Do not position two or more DNA Engine Tetrad 2 cyclers or other thermal cyclers so that the hot exhaust air of one blows directly into the air intake vents of another e Make sure the DNA Engine Tetrad 2 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 Tetrad 2 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 9 nearby equipment running window blinds open lights on Run a typical protocol for 30 minutes to warm up the DNA Engine Tetrad 2 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 trou bleshoot the problem Some experimentation may be required to determine the best solution when mor
47. RAD e 1 800 424 6723 e www bio rad com 5 5 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 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 Bio Rad Technical Support for details 4 Beep Modifies a temperature step or ramp step so the instrument will beep when the target temperature is reached Types of Programs There are two types of Tetrad 2 programs basic and advanced Basic programming fea tures a graphical interface and a graphical representation of the program steps Advanced programming features a text based interface and a descriptive listing of the program steps Program GRAPHH LJ Lid Mode constant 100 00 Program 4pvs1 Lid Control Mode Constant at 100 C 1 Incubate at 34 0 C for 00 01 00 2 Incubate at 92 0 C for 00 00 30 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 End 29 m Add St ur Control Mode carc E Step After ep after ontrol Mode Temp
48. T password protected to a password protected folder Copying from a password protected folder to a password protected folder will prompt you for the Destination folder password Moving a file from a password protected folder to a folder that is NOT password protected will prompt you for the From folder password Moving a file from one password protected folder to another password protected folder will prompt you for the From folder and then the Destination folder passwords Moving a file from a folder that is NOT password protected to a password protected folder will prompt you for the Destination folder password Saving a password protected file will prompt you while clicking Save or lt Save Run gt if the file resides in a password protected folder Save as does not prompt for a password Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 19 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual 5 20 The Graphical Programming Window Before we begin entering program steps let s explore the graphical programming window The graphical programming window displays the steps of 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
49. TS IF YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT OR ANY ACCOMPANYING AGREEMENTS DO NOT OPERATE THE CYCLER OR INSTALL COPY OR OTHERWISE USE THE SOFTWARE PRODUCT IF YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT YOU MAY RETURN THE CYCLER AND SOFTWARE PRODUCT TO BIO OR THE AUTHORIZED SELLER FROM WHICH YOU PURCHASED THE CYCLER AND SOFTWARE PRODUCT BEFORE OPERATING THE CYCLER INSTALLING COPYING OR OTHERWISE USING THE SOFTWARE PRODUCT FOR A FULL REFUND OF THE PURCHASE PRICE THEREOF IN ADDITION BY INSTALLING COPYING OR OTHERWISE USING ANY UPDATES OR OTHER COMPONENTS OF THE SOFTWARE PRODUCT THAT YOU RECEIVE SEPARATELY AS PART OF THE SOFTWARE PRODUCT YOU AGREE TO BE BOUND BY THIS AGREEMENT AND BY ANY ADDITIONAL LICENSING TERMS THAT ACCOMPANY SUCH UPDATES IF YOU DO NOT AGREE TO THE ADDITIONAL LICENSE TERMS THAT ACCOMPANY SUCH UPDATES YOU MAY NOT INSTALL COPY OR OTHERWISE USE SUCH UPDATES BUT MAY RETURN SUCH UPDATES TO BIO FOR A FULL REFUND OF THE PURCHASE PRICE THEREOF IF ANY Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com B 1 M DNA Engine Tetrad 2 Thermal Cycler Operations Manual B 2 1 SCOPE OF AGREEMENT 1 1 No License to Trademarks No license is granted hereunder in connection with any trademarks or service marks of BIO or its suppliers 1 2 This AGREEMENT does not effect any transfer of title in the SOFTWARE PRODUCT 1 3 The equipment and instrumentation included in the CYCLER are not specifically w
50. Tetrad 2 program They concern how your steps should be implemented These decisions involve the following e Temperature control mode e Lid control mode e Temperature ramping rate advanced mode only e Temperature hold time e Temperature time extend advanced mode only e Temperature increment advanced mode only e Temperature gradient available with the 96 well Alpha unit only e Outside of the example protocol other considerations Choosing a Temperature Control Mode The DNA Engine Tetrad 2 cycler can control incubation temperature in two possible ways each of which has different implications for the speed and accuracy of sample heating These include Calculated Control and Block Control Calculated Control When using calculated control the DNA Engine Tetrad 2 cycler estimates sample tem peratures 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 control Since the sample temperature will always lag behind the block temperature the DNA Engine Tetrad 2 cycler can adjust the block temperature to bring samples of a specific volume in a specific vessel type to programmed temperatures This is done through optimized overshoots of the block temperature by a few degrees for a few seconds which bring s
51. Under no circumstances should a customer attempt service of a DNA Engine Tetrad 2 cycler or an Alpha unit as this will result in a voided warranty and may not result in complete problem resolu tion The only exception would be minor maintenance of the units such as removal of vent blockage or routine cleaning as outlined in Chapter 9 10 2 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Troubleshooting Mi Table 10 1 Error Messages The error codes listed in the table below correspond to the error codes returned in the DNA Engine Tetrad 2 cycler s lt Error Log gt The remote error codes listed corre spond to the error codes returned when the DNA Engine Tetrad 2 cycler is operated in remote mode see the lt Remote gt section in Chapter 8 for more information on remote mode Note The DNA Engine Tetrad 2 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 protocol If the message fails to reappear proceed as usual Remote EA ftom O 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 auto matically during and after the run and you will be notified if any per sistent problem is found An irregularity was detected in the block s left temperature sensor The sensor will be checked auto mati
52. Using This Manual This manual contains instructions for operating your DNA Engine Tetrad 2 cycler safely and productively Chapter 2 acquaints you with the physical characteristics of the DNA Engine Tetrad 2 cycler Chapters 3 4 present the basics of installation and operation for the DNA Engine Tetrad 2 cycler Chapters 5 6 and 7 describe the creation editing and running of programs Chapter 8 outlines the software utilities Chapter 9 explains the proper maintenance of the DNA Engine Tetrad 2 cycler Chapter 10 offers troubleshooting information for the DNA Engine Tetrad 2 cycler Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Introduction W Important Safety Information Safe operation of the DNA Engine Tetrad 2 cycler begins with a complete under standing of how the instrument works Please read this entire manual before attempting to operate the DNA Engine Tetrad cycler Do not allow anyone who has not read this manual to operate the instrument 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 guidelines at the beginning of this manual on pages iv and v and exercise all precautions outlined in them i Warning The DNA Engine Tetrad 2 cycler can generate enough heat to figs 2 1 and 2 4 for locations Obstructing air vents can lead to overheating and slightly enhanced risk of electrical shock
53. ages 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 7 7 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual 7 8 Terminating a Run To terminate a run before its completion select the appropriate block from the block selec tion menu in the Status window and select lt Stop gt Alternatively select the lt Command gt menu in the Status window and then select Stop and either lt All gt to terminate all pro grams running 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 Pause Alternatively select the Command menu in the Status window and then select Pause Resume and either Pause All to pause all programs running on all blocks or select the desired block from the drop down list To resume a run select Resume from the bottom of the Status window or select Command lt Pause Resume gt and either Resume All or select the desired block from the drop down list Skipping a Step To skip to the next step in a running program select Skip at the bottom of the Status window A confirmation screen will ask you to confirm the skip select Yes to skip to the next step in the protocol d
54. al Deleting a Program e Select Programs in the Status window Select Delete from the drop down menu Folder Program Listing of ADVH1 Temperature Control Mode Calculated al Lid Control Mode Constant at 100 C 1 Incubate at 34 C for 00 01 00 Beep on Target 2 Incubate at 32 C for 00 00 30 Beep on Target adie 45 D or 00 03 00 Exit 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 convenient 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 verify the deletion Are You Sure You Want to Delete ADV 1 i If you wish to delete the program select Yes If you want to keep your newly cre ated program select No 6 10 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Managing and Editing Programs W Moving a Program e Select Programs in the Status window e Select Move from the drop down menu From Folder Move Program GHAPHZI 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
55. all control method has been 800 4BIORAD in the US or switched to Calculated Mode Canada or your local distributor Results may have been affected outside the US or Canada The power supplytemperature is Please observet he following somewhat higher than normal guidelines 1 place instruments at The power supply sensor will be least 10 cm apart 2 avoid checked and you will be notified placing instruments in any loca if any problem is found tion where hot air might enter the intake vents 3 place instruments on a hard surface with no debris or paper underneath 4 clean all PSO CAF air vents and Alpha unit fins of dust and debris 5 avoid running the instrument in areas with ambient temperatures above 25 C Continue to use the instru ment If problem reoccurs please call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 5 F C E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Remote E Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 6 PSO PT t s SF The power supply temperature was higher than normal To prevent instrument damage the program was automatically stopped Overheating may be caused by inadequate airflow or by a problem with the power supply An irregularity was detected in a power supply temperature sensor The sensor will be checked and you will be n
56. amples to the desired temperature more quickly Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 9 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual 5 10 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 bring samples to the desired incubation temperature rapidly 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 DNA Engine Tetrad 2 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 temperature 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 se
57. ams 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 involves 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 selecting the lt Programs gt with the mouse Drop down submenus appear including Open New Folder Copy Move and Delete e Select New An additional menu appears allowing you to choose Advanced Mode or Basic Mode e Select Basic Mode Program TEMPLATE I Lid Mode CONSTANT e 100 00 Add Step after Control Mode carc Grad GoTo 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 15 B DNA Engine Tetrad 2 Thermal Cycler 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 temper
58. and fire Warning Do not block the DNA Engine Tetrad 2 cycler s air vents see Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 1 3 Layout and Specifications DNA Engine Tetrad 2 Cycler Front View 0000 cece eee eere 2 2 DNA Engine Tetrad 2 Cycler Control Panel 0 ooooomcorrnnn 2 2 DNA Engine Tetrad 2 Cycler Back View ooooooommmmmnmnnnnaan 2 3 DNA Engine Tetrad 2 Cycler Bottom View ooooooommmmmmmmmnmmnmmmnmnmnm 2 3 Compatible Alpha Units 0cocccccc enhn nnn 2 4 DNA Engine Tetrad 2 Specifications llsseeeere 2 5 Gradient Specifications 96 Well Alpha unit only lllllleeee 2 5 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual DNA Engine Tetrad 2 Cycler Front View Figure 2 1 Alpha unit bay Air intake vents Display screen Numeric keypad DNA Engine Tetrad 2 Cycler Control Panel Figure 2 2 1 4 VGA display screen Cursor keys Keypad 2 2 Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com Layout and Specifications W RS 232 port DNA Engine Tetrad 2 Cycler Back View Figure 2 3 Power cord jack Ethernet port Fuses Power switch 2 3 remote command Air exhaust vent Air intake vents
59. ant at 100 C End Entering a Temperature Step While the program ADV 1 is a bona fide Tetrad 2 program it has no utility A run of this program will finish immediately after its start because there are no temperature commands or incubation times to constitute an actual run Recall 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 1 92 C for 30 seconds 5 28 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W 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 B 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 click the left mouse button ONCE Double clicking 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 Cancel 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 Temp Temperature Step Step 1 Temperatur
60. armful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits Note Regarding FCC Compliance Although this design of instrument has been tested and found to comply with Part 15 Subpart B of the FCC Rules for a Class A digital device please note that this compliance is voluntary for the instrument qualifies as an Exempted device under 47 CFR 15 103 c in regard to the cited FCC regulations in effect at the time of manufacture vi Documentation Conventions Before describing the various features of the DNA Engine Tetrad 2 cycler let s define some common ground conventions 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 1 gt gt and lt lt LEFT gt gt e lt gt will be used to indicate windowed menu items or buttons such as lt PRO GRAMS gt lt RUN gt and lt UTILITIES 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 synonymous with click on point and click and any phraseology implying selection of menu or option items with the
61. arranted under this AGREEMENT and any warranties related to the equipment and instrumentation are provided specifically with the equipment and instrumentation of the CYCLER 2 SOFTWARE PRODUCT LICENSE GRANT 2 1 BIO hereby grants LICENSEE a limited non exclusive license to a Use the SOFTWARE PRODUCT including THIRD PARTY SOFTWARE only on the CYCLER b Use the MANUAL and other documentation in support of LICENSEE s use of the SOFTWARE PRODUCT c Make one 1 copy of the SOFTWARE PRODUCT for backup purposes only provided that such backup copy is a complete copy containing all copyright and trademark notices and any other restrictive property legends of BIO that appear on and in the SOFTWARE PRODUCT as originally provided to LICENSEE by BIO d Make a one time permanent transfer of the license granted herein and copies of the SOFTWARE PRODUCT and THIRD PARTY SOFTWARE directly to a third party end user in connection with the sale of the CYCLER to such third party end user provided that i the SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are transferred in their entirety to the third party including all component parts along with all associated media and printed materials including this AGREEMENT as originally received by LICENSEE ii LICENSEE does not retain any copies of the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE or any portion thereof after transfer thereof to the third party and iii the third party agrees in writing to co
62. ature 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 Mode Selection xl Temperature Control Mode v Calculated Block l In Sample Probe Lid Control Mode v Constant F Tracking Tor Power Lid Parameters OK 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 Control Parameters gt The Lid Constant window will appear Lid Constant xl Maintain lid temperature at HE Turn off lid when block temperature drops below E OK 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 5 16 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W e 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 b
63. 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 increment and goto steps Contact Bio Rad Technical Support for details If a ramp rate is not programmed the default will be at maximum Choosing a Temperature Hold Time Because of the calculated melting temperature T4 of a DNA hybrid DNA poly merase 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 11 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 pro grammable hold time is 18 hours with the exception of a forever incubation Choosing a Thermal Gradient Molecular biology labs routinely optimize annealing and denaturing temperatures for thermal cycling reactions Optimization is critical but not always easy The Tm melting temperature of an oligonucleotide can be estimated using an empirically derived correlation which considers a combinat
64. cally during and after the run and you will be notified if any per sistent problem is found An irregularity was detected in the block s right temperature sensor The sensor will be checked auto matically during and after the run and you will be notified if any per sistent problem is found An irregularity was detected in one or more of the block s tem perature sensors The sensor was checked and the resultes show an inaccuracy in block temperature measurement Your program was automatically stopped The heated lid is not preheating correctly Your samples have remained at room temperature and your program was never started Call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada No action is required at this time No action is required at this time No action is required at this time Call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Recover samples and call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 3 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Remote EA fon O HS PS SF PT Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 4 The heat sink temperature is reg istering somewhat higher than normal Power levels will be scaled back slightly until the heat si
65. ce 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 goto step This goto step cannot be the first or only step in the protocol If there is a goto step blue arrow in the displayed protocol Select the goto step 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 Select lt GoTo gt A small red arrow will appear under step 3 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 Select the number of cycles and delete using the backspace key Enter 29 from the numeric keypad and click once to accept the change Program GRAPHH1 LJ Lid Mode constant 100 00 Step 3 gradient step Step 4 Add Step after Control Mode carc goto step Temp Grad 3 Forever Delete Step Save Save As Save Hun Cancel Our program will now run with 30 cycles Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 25 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Entering a Forever Incubation Step 5 of our p
66. ct Advanced Mode Program Listing of ADV 1 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 DIC TI y la 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 steps in the currently highlighted program appears near the bottom of the window 6 2 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 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 ADVH1 Lid Control Mode Constant at 100 C 1 Incubate at 34 0 C for 00 01 00 Beep on Target 2 Incubate at 32 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 ale El Cr Step After Temp Gradient GoTo Lid Delete Step SavesHun 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
67. d see the Tube Microplate and Sealing Selection Chart including Hard Shell thin wall microplates Hard Shell microplates feature a skirt and deck molded from a rigid thermostable polymer that completely 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 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 4 7 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual 4 8 Sealing Sample Vessels To avoid changing the concentration of reactants steps must be taken to prevent the evaporation 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 adequate 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 Chill out liquid wax may be used to seal sam ples Use only a small amount of oil or wax 1 3 drops 15 50 pl are usually sufficient Include this volume in the total volume when setting up a calculated control protocol se
68. d 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 over the thumbwheel as the lid closes over the sample block A click signifies that the Hot Bonnet lid s latch has engaged Selecting the Correct Sample Vessel The DNA Engine Tetrad 2 cycler s wide variety of interchangeable Alpha units affords great scope in choosing sample vessels Keep in mind that differences in tube and plate com position 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 Bio Rad offers a full range of tubes and microplates man ufactured 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 as shown on a DNA Engine thermal cycler 4 6 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Operation M 0 5 ml Tubes Thick walled 0 5 ml 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 0 5 ml tubes specifically designed for thermal cycling The higher qualit
69. 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 Fractional degrees are not accepted Recall that in our example protocol the gradient step should specify a range of 45 C to 65 C and a duration of 3 minutes e Position the cursor text pointer in the higher limit temperature field and select by clicking once e Use the backspace key on the numeric keypad to delete the higher tem perature 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 e 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 clicking once 5 24 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W Use the backspace key to delete the current value if it is not 03 Enter 03 from the numeric keypad and click once to accept the change Position the cursor text pointer in the time second field of step 3 and select by clicking once Use the backspace key to delete the current value if it is not 00 Enter 00 from the numeric keypad and click on
70. e c Beep on Target Hrs Hin Sec Time LIE JE Forever r Options Increment Temp Set Parameters Extend Time Set Parameters FT Slow Ramp Set Parameters OK Cancel The Temperature Step window presents the operator with a number of tem perature 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 avail able 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 incuba tion 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 e Position the cursor over the Temperature field and select e Enter 94 from the numeric keypad into the field Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 29 HM DNA Engine Tetrad 2 Thermal Cycler Operations Manual e Similarly enter 1 in the Time Min field Additionally we want the cycler to beep after reaching the target temperature e Select Beep on Target At this point in the Temperature Step window all selections have been made according to our protocol e Select lt OK gt In the advanced programming window the program listing now appears as follows Temperature Control Mode Calculated L
71. e Gradient Calculator available from the Tools drop down menu in the Status window Since our oligonucleotide annealing temperature is not optimized we will replace our annealing step with a gradient step We will optimize in the range of 45 C to 65 C Your written program should now appear as follows Raw program Use calculated temperature control mode Use constant lid control mode at 100 C Y 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 5 12 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W Beyond the Example Protocol Other Considerations In addition to the above considerations you can also include other protocol varia tions 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 In addition some protocols can include a sustained incubation at sub ambient tem peratures to preserve the integrity of the products We will choose an initial incubation at 94 C for 1 minute before cycling and a final incubation of the sample at 10 C forever Your writ
72. e 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 paraffin waxes solidify at room temperature The wax can then be pierced with a micropipette tip 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 0 The samples are usually pure enough for analysis without an extraction Chill out liquid wax available from Bio Rad is an easy to use alternative to oil This puri fied 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 Bonnet s heated inner lid maintains the air in the upper part of sample ves sels 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 fi
73. e Tetrad 2 Thermal Cycler Operations Manual Entering Program Steps Lii ruoare rir uan dea aia 5 14 The Status WINDOW ses eiieeii Sd Rut rne Dern te icio UR RERO este o dog 5 14 Entering a Program Using Graphical Mode Llsuusss 5 15 Using the Mode Selection Window oooooocooconrooo mm 5 16 Using the File Save As Window cee eee ees 5 17 Folder PaSSWOrdS PP aa ed ar wie Ciera ane ae 5 19 The Graphical Programming Window o oococcccooocc eee eee 5 20 Selecting a Step inca sede Qc rae ty Ree RC e D E t at 5 20 Editing Step Parameters pcia 4 Wa e e 5 20 Deleting a Step EDD 5 21 Adding a Step TP 5 21 Entering a Temperature OD im mr uox Ro b do ea ede Rok 5 21 Enterma a Gradient Step dc redo za E edes uu pad 5 24 Entering a Goto Ste aes dira edu doe R S OA ra CR de ease Rl d 5 25 Entering a Forever Incubation o oooooooooommooooos 5 26 Entering a Program Using Advanced Mode o o ooccocoocoo eee ee eee 5 27 Entering a Temperature Step 42 229 adem Rad EE LAquee A 5 28 Entering a Gradient Step esos rra RR Ra CR RC R Re OE RUE 5 81 The Extend Time Option o ooocococoooo eee 5 33 Entering a Goto Stes uiae Sirio Io A cR CR e po RR Ra d 5 34 Entering a Lid Control Step asiarobarreoriocpri ttita niaes 5 35 The Slow Hamp ODIO uaa ira HR 5 35 The Increment Temp Option 00 cece eee 5 36 The Moto Alpha Unit 0 ees 5 38 5 2 Tech Suppor
74. e instrument in areas with ambient temperatures above 25 C Continue to use the instru ment If problem reoccurs please call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada No action required at this time Troubleshooting Mi Remote SA ation O The internal fan is not providing Call 800 4BIORAD in the US or adequate cooling for the instru Canada or your local distributor ment outside the US or Canada An irregularity has been detected No action required at this time in a heated lid sensor The heated lid has been temporarily turned ES off The sensor will be checked and you will be notified if any problem is found M An imbalance has been detected Call 800 4BIORAD in the US or LRPI in the sample block There may be Canada or your local distributor a problem with a Peltier module outside the US or Canada A memory fault has been found Call 800 4BIORAD in the US or Stored programs may be affected Canada or your local distributor outside the US or Canada All available memory has been Please delete unused programs filled or call 800 4BIORAD in the US or Canada or your local distrib utor outside the US or Canada A possible fault was detected in Check probe and connections the in sample probe The thermal If problem reoccurs please c
75. e plug will disengage Pull the plug straight out to expose the fuse figure 9 1B 3 Remove all fuses and replace them with new ones it is often impossible to deter mine 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 Reconnect the power cord Figure 9 1 A How to pull out the fuse plug B Location of the fuses in the opened plug o EE o Fuse Plug Screwdriver Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com 9 3 10 Troubleshooting Sources of Problems arica 10 2 System Problems essa Qoae a a o De Dar aca 10 2 Error Messages ulis uses kidi cia REUS aaa 10 3 Problems in Power Up 2 00 cece eee eee 10 8 Problems with System Performance llli 10 9 Problems with an Alpha Unit 0 0 00 cee eee es 10 10 Problems Related to Protocols 0000 cece eee eee ee eens 10 10 Problems due to Environmental Conditions Setup and Maintenance 10 13 10 1 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Sources of Problems The DNA Engine Tetrad 2 cycler is designed to handle even the most stringent thermal cycling requirements A major strength of the DNA Engine Tetrad 2 cycler is its flexibility which allows multifaceted and demanding cycling protocols to be implemented with the greatest of exper
76. e than one cause is involved After taking steps to solve the problem verify that the temperature of the air entering the air intake vents has been lowered using the procedure outlined above Requirements for Robotics Installations Robotics installations require special attention to airflow and air temperature Typically in these installations DNA Engine Tetrad 2 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 temperature 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 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 3 5 3 6 DNA Engine Tetrad 2 Thermal Cycler Operations Manual 384 Well Microplate Specifics Some users find that a 384 well microplate can be difficult 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
77. econds 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 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 Temp 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 23 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Step two of our protocol should now specify a temperature incubation of 92 0 for a duration of 00 30 Step 2 Program GRAPH 1 DJ Lid Mode CONSTANT 100 00 temperature step Add Step after Control Mode carc Grad GoTo Forever Delete Step Save Save As Save Hun Cancel Entering a Gradient Step Step three of our program is a gradient step designed to determine the optimal annealing temperature of our oligonucleotide If step three of the displayed protocol is not a gradient step two black lines Select step two e Select lt Grad gt A new gradient step should now appear as step three with the default higher temperature limit of 65 C and the
78. elow 30 C e Enter 30 from the numeric keypad e Select lt OK gt We have returned to the Mode Selection window e 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 Delete Step gt lt Save gt lt Save As gt or lt Save Run 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 graphical program with the default name TEMPLATE be run This restriction is designed to ensure that programs are appropriately saved e Select lt Save As gt New Folder Program Name TEMPLATE Edit Hame OK Cancel 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 field The lt New Folder gt button creates a new folder in which to store your new program Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com
79. ementation of the Tetrad 2 programs and the instant incubation feature Using the Instant Incubation Feature The DNA Engine Tetrad 2 cycler can also be quite useful as an instant constant temperature incubator with a range from 5 0 C to 105 C 4 C to 100 C with the Slide Chambers Alpha unit This feature can be used for performing ligations digestions etc or with slides per forming overnight humidified hybridizations Note The Slide Chambers Alpha unit can be used as a humidified chamber for steady state incubations e g hybridizations color development reactions To humidify a block push one laboratory tissue into the bottom slot and inject 1 ml of deionized water onto it See the Slide Chambers Alpha Unit Operations Manual for complete instructions To initiate an instant incubation from the DNA Engine Tetrad 2 cycler s Status window Programs Utilities Command View steals Block 14 30 y Block 14 30 bd UserName GUEST El Temperature Time Block 228 C Step 00 00 00 S ample 228 C Remaining 00 00 00 Lid 21 9 C Cycle D Instant Stop Pause Skip e Select Instant at the bottom of the window or from the Command menu 7 2 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Program MYFIRST user GUEST y r Select Blocks Select All Clear Indicates Block Type Used for Calc Mode DK Cancel Running Programs W
80. en affected in any way The elapsed time displayed under the graph will give the true elapsed time of the protocol Select Status to return to the Status window 7 6 Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com Running Programs W The Status window will display Block Sample and Lid temperatures corre lating to the program running on the block chosen in the block selection menu These temperatures represent real time readings and correspond to the values represented 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 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 all 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 from the drop down list Status All Blocks xl Block Block Program Time Time Name Status Running Remaining Elapsed User 1 96 Active MYFIRST 02 10 34 00 06 14 GUEST Completed with No 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 mess
81. er insert in order to properly and safely remove the instrument from its packaging Always enlist the help of another individual when moving or lifting the DNA Engine Tetrad 2 cycler In order to lift the instrument grasp the DNA Engine Tetrad 2 cycler from underneath the chassis placing your hands on either side of the DNA Engine Tetrad 2 cycler in between the feet of the instrument and lift Do not insert the Alpha units until the instrument is located in its final place Packing Checklist After unpacking the DNA Engine Tetrad 2 cycler check to see that you have received the following e One DNA Engine Tetrad 2 base e Four spare fuses e One power cord e One external mouse device e DNA Engine Tetrad 2 Peltier Thermal Cycler Operations Manual this document e Unpacking Instructions for the DNA Engine Tetrad 2 Thermal Cycler If any of these components are missing or damaged contact Bio Rad Laboratories to obtain a replacement Please save the original packing materials in case you need to return the DNA Engine Tetrad 2 cycler for service Setting Up the DNA Engine Tetrad 2 Cycler The DNA Engine Tetrad 2 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 220 V electrical outlet With the machine switched off mount the Alpha units See the Insta
82. fying 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 shorter 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 Designing a New Program The first step in designing any program is the translation of your experimental protocol into Tetrad 2 program steps We suggest writing all steps until you are rea sonably comfortable with Tetrad 2 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 a 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 Tetrad 2 program design and be able to enter steps directly from experimental protocols However we strongly suggest following these steps
83. g In 1 Declaration of Conformity 00 00 eee DoC 1 Explanation of Symbols gt gt D CAUTION Risk of Danger Wherever this symbol appears always consult note in this manual for further information before proceeding This symbol identifies components that pose a risk of personal injury or damage to the instrument if improperly handled CAUTION Risk of Electrical Shock This symbol identifies components that pose a risk of electrical shock if improperly handled CAUTION Hot Surface This symbol identifies components that pose a risk of personal injury due to excessive heat if improperly handled Safety Warnings and Guidelines gt e BPE PEE Warning Warning Warning Warning Warning Caution Caution When removing an Alpha unit from a DNA Engine Tetrad 2 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 DNA Engine Tetrad 2 cycler before reading this manual can constitute a personal injury hazard Only qualified laboratory per sonnel trained in the safe use of electrical equipment should operate these machines Do not open or attempt to repair the DNA Engine Tetrad 2 cycler any Alpha unit or any accessory to the cycler Doing so will void your war ranties and can put you at risk for electrical shock Return the DNA Engine Tetrad 2 cycler to the factory US customers or an autho
84. g insertion point will appear in the temperature field See the Selecting a Step and Editing Step Parameters sections on page 5 21 for additional selection and editing options Use the backspace key on the numeric keypad to delete the current tem perature if it is not 94 0 C Enter 94 from the numeric keypad Click once to accept the change Position the cursor text pointer in the time minute field of step 1 and select by clicking once Use the backspace key to delete the current value if it is not 01 Enter 01 from the numeric keypad and click once to accept the change Position the cursor text pointer in the time second field of step 1 and select by clicking once Use the backspace key to delete the current value if it is not 00 Enter 00 from the numeric keypad and click once to accept the change Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W Step one of our protocol now consists of a temperature step with an incubation tem perature of 94 0 and a duration of 01 00 Program GRAPH DI Lid Mode constante 100 00 Step 1 temperature step Add Step after LJ Control Mode carc Temp Grad GoTo Forever Delete Step Save Save As Save Hun Cancel Recall that the maximum programmable temperature is 105 0 C and the minimum programmable 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 s
85. ha Units o occoccoccc ee 4 4 Installing an Alpha Unit duae y eed a arm RR ROS de ace e ea ee oe 4 4 Removing an Alpha DII scese a IA bxc lo dom i o 4 5 Opening an Alpha Units 2a ckc ouo aC Dd ROI AAA AAA 4 6 Closing an Alpha UME 2o uas tieu eub pav b X sate aac dudar ai a atas da beeen 4 6 Selecting the Correct Sample Vessel lseeeeeeeese 4 6 0 5 ml TUD6S 5 a aeu m ac are aci matin di sry ERU NSA Sb are dieran ari dal af 4 7 Thin Walled vs Thick Walled Tubes 0 0 eee tect essen 4 7 0 2 ml TUDES i usezam chm Rack heme aaa AUC RU s 4 7 Microplates od sed wee 9 de eRe ia e aet c 4 7 Sealing Sample Vessels 000 cee eee nnn 4 8 Sealing with Oil or Wax scs5 c0e wave ee noes aoe Oe ash A ede es bas wd 4 8 Sealing with the Hot Bonnet Lid 2cacadctous baddadteennaveadeuedsaas 4 8 Adjusting the Hot Bonnet Lid s Pressure ooooomoo ooo 4 9 Loading Sample Vessels into the Block ree 4 10 Using Oil to Thermally Couple Sample Vessels to the Block 4 10 Appendix 4 A Tube Microplate and Sealing System Selection Chart 4 12 Appendix 4 B Safety Warning Regarding Use of 35S Nucleotides 4 13 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Turning the DNA Engine Tetrad 2 Cycler On cycler turned on electrical arcing can result Read the safety warning regarding electrical safety on page iv of this manual before
86. he thermal gradient option is only available when using 96 well blocks Transferring Program Files During the initial boot up sequence of the DNA Engine Tetrad 2 cycler see the Turning the DNA Engine Tetrad 2 Cycler On section in Chapter 4 several options are available for transferring program files Options 2 Send Files and 3 Receive Files can be used to transfer all program files in all folders on one DNA Engine Tetrad 2 cycler to another DNA Engine Tetrad 2 cycler To perform a file transfer from one DNA Engine Tetrad 2 cycler to another 1 Use a standard RS 232 null modem cable to connect the DNA Engine Tetrad 2 cyclers via their RS 232 ports Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 8 7 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 2 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 3 During the boot up sequence of the sending instrument select the Send Files option by entering 2 on the numeric keypad 4 To execute the file transfer select Proceed 48 Exit when completed by entering 1 on the numeric keypad of the sending instrument Once the transfer is com plete the TRANSFER COMPLETE message will be displayed and the instrument will proceed with the boot sequence 8 8 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 9 Maintenance
87. i lt Control gt lt Remote gt To operate a DNA Engine Tetrad 2 cycler in remote mode i e from a desktop computer select lt Remote gt from the lt Control gt menu The Remote Mode window will appear on the screen Remote Mode ES Syntax Checking Auto Remote Clear Select the Syntax Checking check box to filter out any commands that do not conform to the PTC Remote Command Set syntax Contact Bio Rad Technical Support for the latest syntax of the remote command set Attach a null modem serial interface RS 232 cable to the RS 232 port connector on the back of the DNA Engine Tetrad 2 cycler Connect to either COMM1 or COMM on a PC or the serial port on a Mac The cycler is now ready to be run in remote mode Select the Auto Remote check box to direct the DNA Engine Tetrad 2 cycler to auto matically enter remote mode upon subsequent power up This feature is particularly useful if you consistently control the DNA Engine Tetrad 2 cycler using a desktop computer It eliminates the need to select Remote after every power up To disable the auto remote feature deselect the check box see the warning below The cycler will now power up in standard operation mode Important No protocols can be running when entering 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 prot
88. id 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 temperature e Position the cursor over the appropriate insertion point if not highlighted already and select Note The appropriate insertion point would be selected by highlighting the first step Insertion will occur AFTER this step e Select lt Temp gt e Position the cursor over the Temperature field and select e Enter 92 from the numeric keypad into the field e Similarly enter 30 in the Time Sec field Additionally we want the cycler to beep after reaching the target temperature e Select Beep on Target At this point in the Temperature Step window all selections have been made according to our protocol e 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 5 30 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W 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 p
89. imental 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 related to protocols e Problems due to environmental conditions setup and maintenance When troubleshooting a difficulty it is advisable to have the following pieces of information available should it become necessary to contact Bio Rad Laboratories 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 chapters In the Status window the View submenu allows the user to view the Error Log 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
90. implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA 05184 rev E Table of Contents Explanation of Symbols ooococoo lll iv Safety Warnings and Guidelines 00 0 cece ees iv Electromagnetic Interference and FCC Warning ooooooccoccooronn ees V Documentation Conventions o oococooo lr vi Chapter 1 Introduction o o oocococo cono 1 1 Chapter 2 Layout and Specifications llle 2 1 Chapter 3 Installation z 4 is pee RR RR EL Rm em md ET a 3 1 Chapter 4 Operati0N ooocoococooo lh 4 1 Chapter 5 Creating Programs seeeeeeseeeeee eere 5 1 Chapter 6 Managing and Editing Programs leeren 6 1 Chapter 7 Running Programs sese 7 1 Chapter 8 Using the Utilities ooooooooooommormmmo o 8 1 Chapter 9 Maintenance lesse 9 1 Chapter 10 Troubleshooting axxo nec Ron eee eve E ERREUR SG 10 1 Appendix A Warranties o oooooconoooonn rn A 1 Appendix B End User and License Agreement o ooocccccccocoo B 1 Appendix C Factory Installed Protocols 0 0 00 cece eee ees C 1 Index tee ee ae eae Seed ae Ra ee wee A ee ha te he o
91. ine Tetrad 2 cycler can use calculated mode to compensate accordingly The cycler defaults to Calculated mode Refer to the section Choosing a Temperature Control Mode below for additional information 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 DNA Engine Tetrad 2 cycler defaults to Constant Refer to the Choosing a Lid Control Mode section below for addi tional information Temperature step This sets incubation temperature and duration The DNA Engine Tetrad 2 cycler 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 Slide Chambers 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 from 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 Moto Alpha unit to automatically open o
92. ine or front end software e Select Update Soft from the Utilities menu to access the HTTP Update Software Utility window e Select Display Versions to view the available software upgrades e Click on the arrow button to the right of the Select Software Version field to dis play the available versions e Select the software that you wish to download 8 6 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Using the Utilities Mi e Select Update e Aconfirmation screen will appear asking you to confirm your desire to update the software Select Yes 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 Log to view the error log If the update was successfully completed restart the DNA Engine Tetrad 2 cycler e Select About from the Utilities menu to view the currently loaded software version and confirm that the software update has been successfully completed Bio Rad also offers a Software Update Utility Kit which allows users to update the software of any DNA Engine line thermal cycler including the DNA Engine Tetrad 2 using a serial cable and a Windows based PC This kit is available free of charge from Bio Rad Request item number SWU 0200 Gradient Calc The Gradient Calculator allows you to view the temperature of any well or column in the sample block As discussed in Chapter 5 t
93. ing fail safe performance or any other applications or situations in which the failure of the CYCLER or SOFTWARE PRODUCT could lead directly to death personal injury or severe physical or environmental damage HIGH RISK ACTIVITIES Accordingly BIO specifically disclaims any express or implied warranty of fitness for High Risk Activities LICENSEE agrees that BIO will not be liable for any claims or damages arising from the use of the CYCLER or SOFTWARE PRODUCT in such applications or situations Note on Java Support The SOFTWARE PRODUCT may contain support for programs written in Java Java technology is not fault tolerant and is not designed manufactured or intended for use in HIGH RISK ACTIVITIES Sun Microsystems Inc has contractually obligated THIRD PARTY SOFT WARE suppliers to make this disclaimer 5 INDEMNIFICATION LICENSEE agrees to indemnify and hold harmless BIO including its officers directors employees and agents and suppliers from and against any claims or lawsuits including attorney fees that arise or result from any negligent unlawful or unauthorized use transfer or distribution of the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE by LICENSEE 6 TERM AND TERMINATION e Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Appendix B End User Agreement and License M 6 1 Term The term of this AGREEMENT and the license granted hereunder shall com mence upon first use of the CYCLER or first
94. inimize primer dimers Program a hot start into the protocol Check concentrations of compo nents May need to reoptimize magnesium concentration Reoptimize annealing temperature Use List utility to check protocol s temperature control method tem peratures and times Check extraction and purification protocols Add additional purifica tion steps if necessary Check nucleic acid preparations by gel electrophoresis 10 12 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Troubleshooting Mi 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 DNA Engine Tetrad 2 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 sug gested solutions Table 10 3 Environmental Difficulties Problem Cause Action Frequent shutdowns Machine is not receiving enough air Make sure air intake vents are not due to overheating obstructed by dust debris or Frequent Slow Block paper Remove light collections of Cycling HS Overheat dust and debris with damp cloth and HS Overheating Vacuum out heavy collections error messages Remove any papers placed under the machine Position machine at least 10 cm from vertical surfaces
95. ion of DNA length G C content and salt concentration However since the T 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 DNA Engine Tetrad 2 cycler programmable temperature gradient feature allows for optimization of an incubation temperature in a single experiment by analyzing a number of different temperatures 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 exper iment 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 pro grammable temperature is 105 C the minimum programmable temperature is 30 C Note The programmable temperature gradient feature is only accessible if a 96 well Alpha unit s is mounted in the DNA Engine Tetrad 2 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 th
96. ip for recovery of sample from individual wells Microplate 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 Microseal P and P sealing pads are designed for use in applications such as cycle sequencing in which several successive runs may be sealed with the same pad Microseal P and P pads are intended for use with the higher sealing pressures afforded by the Power Alpha units use P pads with the original version of Power Bonnet lids ALP 1296 and ALP 1238 and P pads with newer design Moto Alpha units ALP 2296 and ALP 2238 Each pad may be used for approximately 25 runs 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 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 micro
97. isplayed 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 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 is no Moto Alpha unit 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 Slide Chambers 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Running Programs W Running Multiple Programs One particularly useful feature of the DNA Engine Tetrad 2 cycler is the ability to run sev eral programs at once on different blocks For example in a DNA Engine Tetrad 2 cycler setup with two 96 well Alpha units a gradient for optimizing annealing temperature can be ru
98. istributor outside the US or Canada Call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Troubleshooting Mi Remote A program containing a gradient step can only be run on a 96 well Alpha unit The program entered may only be run in Calculated or Block tem perature control mode Probe mode is unavailable for this type of program For optimum results the temperature control mode has been automatically switched to Calculated control The thermal gradient has not been achieved as quickly as expected This may indicate a possible problem with the Alpha unit The instrument has detected unexpected temperature read ings This may indicate a problem with the Alpha unit A ground fault was detected in an Alpha unit Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com If 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 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Replace fuse on instrument and confirm that instrument is func tional If problem reoccurs please call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada
99. ld 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 Save to ensure that your work is preserved Chapter 6 will explain how to edit the various programming steps to include different parameters and Chapter 7 will instruct you on how to run this program Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 5 37 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Configuring the Tetrad 2 with a Moto Alpha Unit 5 38 The opening and closing of the robotic lid can be accomplished either manually by pressing the blue button on the lid or set through the software The electronically controlled heated lid features software configurable lid pressure and lid opening angle settings Instructions for programming the Moto Alpha unit s lid are found in the document Remote Control Command Sets This document is available from the Bio Rad website Lid Pressure The pressure exerted by the Moto Alpha unit s inner lid must be adjusted to fit a particular type of reaction vessel Once set the same pressure is maintained until the lid is readjusted for a different type of vessel Lid pressure is set either through the front panel of the cycler or
100. ler s connectors When the handle is in the down position the Alpha unit is locked into place 4 4 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Operation W Removing an Alpha Unit cycler turned on electrical arcing can result Read the safety warning at the beginning of this manual on page iv regarding electrical safety when inserting or removing an Alpha unit 1 Caution Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2 1 Turn off the DNA Engine Tetrad 2 cycler 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 outward from the center of the cycler The electrical connectors of the Alpha unit and the DNA Engine Tetrad 2 cycler are now disen gaged and there is little danger of electrical shock Slide the Alpha unit from the center of the DNA Engine Tetrad 2 base about 3 cm 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 as shown on a DNA Engine Dyad thermal cycler A Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 4 5 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 upwar
101. lling an Alpha unit section in Chapter 4 cycler turned on electrical arcing can result Read the safety warning at the beginning of this manual on page iv regarding electrical safety when inserting 1 Caution Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2 or removing an Alpha unit Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Installation Mi External Mouse Device Included with each shipment of a DNA Engine Tetrad 2 thermal cycler is an externally attachable mouse which should be attached prior to power up of the DNA Engine Tetrad 2 cycler Usage of the mouse is required to access full function ality of the programming software Underneath the front lip of the DNA Engine Tetrad 2 cycler are two connection ports see figure 2 4 The mouse should be connected to the USB A port To ensure complete compliance with FCC and EMC requirements only a mouse with a ferrite core should be used with the DNA Engine Tetrad 2 instru ment To connect the mouse please follow these steps 1 Verify that the DNA Engine Tetrad 2 cycler is off Wait for 10 seconds to ensure that all fans have stopped rotating 2 Grasping the sides of the DNA Engine Tetrad 2 cycler tilt the instrument back so that the underside of the lip is visible Insert the mouse into the USB A port and push the connector into place 4 Tip the DNA Engine Tetrad 2 cycler back down and power up the system Environmental Requiremen
102. lock 4 10 Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com Operation W Caution If you use oil in the block use only mineral oil Never use silicone oil lt can damage the Alpha unit One exception to this recommendation involves the use of volatile radioactive 35S nucleotides 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 compounds in polypropylene tubes and polypropylene and poly carbonate microplates A second exception involves the use of thick wall 0 5 ml 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 chan Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 4 11 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual Appendix 4 A Tube Microplate and Sealing System Selection Chart The following sample vessels and sealing options are recommended for use with the DNA Engine Tetrad 2 cycler and are available from Bio Rad Key 4 Reaction vessel fits block sealing option fits reaction vessel without modification Reaction vessel sealing option can be cut to fit Bio Rad Thermal Reaction Vessels Sealing Options for Oil Free Cycling Cycler Blocks 60 30 Description
103. mply with all of the terms and con ditions contained in this AGREEMENT including the obligation not to further transfer this AGREEMENT and SOFTWARE PRODUCT Such transfer may not be by way of consignment or any other indirect transfer 2 2 Restrictions LICENSEE agrees that LICENSEE shall not a Separate the components of the SOFTWARE PRODUCT for use by more than one user b Copy the SOFTWARE PRODUCT THIRD PARTY SOFTWARE or the MANUAL except and to the extent provided in Paragraph 2 1 c c Sublicense distribute disclose or transfer the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE in whole or in part to any third party except to the extent as provided in Paragraph 2 1 d and 2 2 d d Sublicense the SOFTWARE PRODUCT or the THIRD PARTY SOFTWARE except in connection with the rental or leasing of the entire CYCLER to a single end user in a bona fide commercial rental or leasing transaction and only if said end user agrees in writing to be bound by the terms of this Agreement Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Appendix B End User Agreement and License M e De compile reverse compile reverse engineer disassemble or modify the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE or any portion thereof in any way f Use the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE or any portion thereof for development of any infringing or derivative works 2 3 Notice The SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are p
104. n 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 before 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 pro tocol should be run and the calculated control parameters for the run if applicable as described below To run preprogrammed or previously created programs see Appendix C Factory Installed Programsp Chapter 5 Creating Programs and Chapter 6 Managing and Editing Programs you must first load them into memory From the Status window e Select Run at the bottom of the window or from the Command menu e From the open program window that appears use the external mouse to select the desired Folder and Program Note the factory programmed protocols are initially stored in the Main folder Double check the program listing to ensure that the listed steps are consistent with the desired program e Select OK You are now presented with the Run Program window Tech Support 1 800 4BIORAD e 1 8
105. n in one Alpha unit while a typical experiment without a gradient can be run in another 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 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 to initiate an independent run Subsequent chapters will review the remaining menu and submenu items particularly the Utility functions Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 7 9 8 Using the Utilities SADOU a RA AAA AAA A A a 8 2 derer EE 8 3 A O 8 3 Config 22 A A A ERES E NEM 8 4 User Names ua uices em ya la d ec ee Sem a ble 8 4 ost Date TIMES sero aua da Eae uates XS udua weds Bue es E ale us lo ac a 8 4 Bay COMGMANONS esse la a Ee ewe as el 8 5 Bay Identification isse A A wae eed 8 5 tuv y nissan is orice eee e td A 8 5 ROOINICE P Qr 8 5 Network iso is nea REA REESE EE ERES a UE RUE S EUR RENE CS qu ees 8 6 Update Soft amp Network ConllQg sss eurer car LE
106. nk temperature returns to normal Adequate airflow is nec essary to prevent the heat sink from overheating The heat sink reached a tempera ture higher than normal and the prgram was automatically stopped Adequate airflow is nec essary to prevent the heat sink from overheating Faults have been detected in sen sors in the heat sink and power supply To prevent overheating the program has been automati cally stopped An irregularity has been detected in a heat sink sensor The sensor will be checked and you will be notified if any problem is found Please observet he following guidelines 1 place instruments at least 10 cm apart 2 avoid placing instruments in any loca tion where hot air might enter the intake vents 3 place instruments on a hard surface with no debris or paper underneath 4 clean all air vents and Alpha unit fins of dust and debris 5 avoid running the instrument in areas with ambient temperatures above 25 C Continue to use the instru ment If problem reoccurs please call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada Please observet he following guidelines 1 place instruments at least 10 cm apart 2 avoid placing instruments in any loca tion where hot air might enter the intake vents 3 place instruments on a hard surface with no debris or paper underneath 4 clean all air vents and Alpha unit fins of dust and debris 5 avoid running th
107. ntration 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 Bio Rad 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 9596 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 the block in an attempt to clean it Severe damage to the block the heated lid and the chassis may result Caution Do not pour any cleaning solution into the block s wells and the heat 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
108. ocol A confirmation screen will ask you to confirm your intent to exit the Remote Mode window Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 8 3 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual lt Config gt lt User Name gt When this item is selected the following screen will appear User Name GUEST GUEST al Add User Name E OK Cancel By selecting Add User Name the virtual keyboard will appear allowing users to enter their name before executing a program In this manner any potential user can deter mine the owner of a block the person running a program as the 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 DNA Engine Tetrad 2 cycler s time Set Date Time x Month Day Year Dale Hrs Hin Sec 23 59 59 Time ro zz pa Cancel Enter the appropriate information in the fields provided and select lt OK gt 8 4 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Using the Utilities Mi lt Bay Configuration gt This utility allows the user to reconfigure the status loop on the DNA Engine Tetrad 2 cycler to power up to four blocks Select the appropriate box to configure each bay Select All will enable the DNA Engine
109. ords Users have the ability to secure their protocols by creating a folder password at the time of folder creation After assigning a name to a new folder the New Folder window displays the lt Edit Password gt button e Select Edit Password The Change Password message box appears To assign a password to a new folder select the Edit button next to the New window The Edit KeyPad window appears Select letters to compose a password for this folder Passwords cannot be longer than twelve characters Select OK to enter the password You will need to verify the spelling of this password by selecting the Edit button next to the Confirm window Again select the same characters to spell out the new password and select lt OK gt You can also password protect or change the password of a folder that has already been created by selecting Folder Password from the Programs gt Folder menu From the Folder Password dialog box select the Folder Password option and then high light the folder of interest Click the Edit Password button If the folder has an existing password you will be prompted to enter the Old and New passwords Confirm the spelling of the new password You must present a password whenever a change occurs to the contents of a folder that is password protected Several instances are listed below Deleting files or folders from a password protected folder Copying from a folder that is NO
110. otified if any problem is found The heat sink reached a tempera ture higher than normal and the prgram was automatically stopped Adequate airflow is nec essary to prevent the heat sink from overheating The heated lid took slightly longer than expected to reach its target temperature There may be a problem with the lid sensor or lid heater Please observet he following guidelines 1 place instruments at least 10 cm apart 2 avoid placing instruments in any loca tion where hot air might enter the intake vents 3 place instruments on a hard surface with no debris or paper underneath 4 clean all air vents and Alpha unit fins of dust and debris 5 avoid running the instrument in areas with ambient temperatures above 25 C Continue to use the instru ment If problem reoccurs please call 800 4BIORAD in the US or Canada or your local distributor outside the US or Canada No action required at this time Please observet he following guidelines 1 place instruments at least 10 cm apart 2 avoid placing instruments in any loca tion where hot air might enter the intake vents 3 place instruments on a hard surface with no debris or paper underneath 4 clean all air vents and Alpha unit fins of dust and debris 5 avoid running the instrument in areas with ambient temperatures above 25 C Continue to use the instru ment If problem reoccurs please call 800 4BIORAD in the US or Canada or your local d
111. plate or tubes 2 Open the Hot Bonnet lid Turn the 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 thumb wheel 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 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 4 9 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual 5 For microplate sealing films or mats that require additional pressure turn the thumbwheel clockwise an extra half turn past the point of initial contact to set an appropriate lid pressure make it hard or impossible to close the lid and puts excessive strain on Caution Do not turn the thumbwheel more than three quarters of a turn This can N the latch holding the lid closed An extra half to three
112. ple 7 9 saving 5 17 5 19 6 8 types 5 6 5 7 advanced 5 7 graphical 5 6 Programming conventions 5 4 Radioactive contamination choosing microplates and tubes 4 13 cleaning 4 14 problem with 35S nucleotides 4 13 solutions to 35S problem 4 13 Ramping rate 2 5 5 11 Receive files 8 8 Removing microplates See Microplates Resuming a run 7 8 Robotics installations requirements for 3 5 Run initiating 7 4 7 5 pausing resuming 7 8 skipping a step 7 8 status 7 6 7 7 Index M Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com In 9 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual terminating 7 8 Run program window 7 5 Running multiple programs 7 9 Running programs 7 4 7 5 S Safety general instructions 1 3 Use of 35S Nucleotides 4 13 Sample vessels ensuring good thermal contact 4 10 loading into block 4 10 sealing reason for 4 8 selection chart 4 12 with Hot Bonnet lid and caps film 4 8 with oil or wax 4 8 selection 4 6 0 2 ml tubes 4 7 0 5 ml tubes 4 7 microplates 4 7 selection chart 4 12 thin vs thick walled tubes 4 7 Use of oil to improve thermal contact with block 4 10 Save Run 7 4 Saving a program 5 17 5 19 Saving edited programs 6 8 Selftest 4 2 Send files 4 2 8 8 Setting machine up 3 2 See also Alpha units installing Skipping a step 7 8 Slide Chambers Alpha unit 2 4 7 2 9 2 In 10 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Specifications 2 5 Slow
113. 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 cannot be opened in basic mode because it contains the step modification option Beep on Target If an advanced program cannot be opened in basic mode the following message will appear This file has options not available to the Basic Mode Editor Do you want to open the File with the Advanced Mode Editor Select lt Yes gt to open the program in advanced mode Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 6 3 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Opening a Program in Basic Mode You can choose to open and edit a graphical program in either the graphical pro gramming window or in the advanced programming window Opening a graphical program in advanced mode is desirable if you wish to add step modification options incubations below 0 C or other programming features not available in graphical pro grams 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 proce dure is similar to that described above for opening a program in advanced mode and is summarized here Select Program
114. quarters of a turn ensures the correct pressure for most types of reaction vessels Some empirical testing may be required to deter mine the optimum pressure required for certain vessels Once this pressure has been determined the thumbwheel position may be marked with a col ored 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 Adjusting 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 instructions e Firmly press individual tubes or the microplate into the block wells Using Oil to Thermally Couple Sample Vessels to the Block With two exceptions see below Bio Rad Laboratories 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 b
115. r ized distributor all other customers if repairs are needed All Alpha unit blocks can become hot enough during the course of normal operation to cause burns or cause liquids to boil explosively Wear safety goggles or other eye protection at all times during operation The DNA Engine Tetrad 2 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 DNA Engine Tetrad 2 base you could receive a serious electrical shock Opening the base will also void your warranty Never remove an Alpha unit from the DNA Engine Tetrad 2 cycler with the power turned on and a program running Doing so can cause elec trical arcing that can melt the contacts in the connector joining the Alpha unit to the cycler Do not attempt to unpack or move the DNA Engine Tetrad 2 cycler alone Doing so could results in personal injury Always enlist the help of another individual when moving or lifting the DNA Engine Tetrad 2 cycler Safe Use Guidelines The DNA Engine Tetrad 2 cycler is designed to be safe to operate under the following conditions e Indoor use e Altitude up to 2000 m e Ambient temperature 5 31 C e Relative humidity 10 90 noncondensing e Transient overvoltage per Installation Category Il IEC 664 e Pollution degree 2 in accordance with IEC 664 Electromagnetic Interference This device complies with Part 15 of the FCC Rules Ope
116. r 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Managing and Editing Programs W Copying a Program e Select Programs in the Status window e Select Copy from the drop down menu From Folder Copy Program To Folder ADVANCED BASIC EXAMPLE 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 users 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 Copy The program now resides in the destination folder e Select Exit to return to the Status window Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 6 9 E DNA Engine Tetrad 2 Thermal Cycler Operations Manu
117. r close only available in advanced programs End step Automatically included this instructs the DNA Engine Tetrad 2 cycler to shut down its heat pump because the program is complete Additional program modifications are available with advanced programs see the Types of Programs section immediately following for more information on advanced 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 incre menting to lower stringency the reaction favors the desired product by creating 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 exe cuted This capability is useful for slowly increasing typically by 2 to 5 seconds per cycle the hold time during an extension step The number of bases that a Tech Support 1 800 4BIO
118. ramp option 5 35 Software update 8 6 Specifications 2 5 Gradient 2 5 See also Gradient Status 7 6 7 7 block cycle and lid temperatures 7 6 time elapsed 7 6 time remaining 7 6 Status all 7 7 Status window 5 14 T Temperature control methods block control 5 10 calculated control 5 9 modifying a program from a different machine 5 10 modifying block control programs 5 10 Temperature hold time 5 11 Temperature increment See Increment temp option Temperature range of Tetrad 2 cycler 2 5 Temperature step advanced program 5 28 5 31 graphical program 5 22 Temperature step widow 5 31 Terminating a run 7 8 Thermal accuracy 2 5 Thermal uniformity 2 5 Time Date setting 8 4 Touchdown 5 5 See also Increment temp option Tracking mode 5 11 Transferring program files 8 8 Index M Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com In 11 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 problems 10 10 system problems 10 2 U Uniformity See Thermal uniformity Update soft utility 8 6 User name 8 4 Utilities about 8 2 network config 8 6 set date time 8 4 update soft 8 6 user name 8 4 V Virtual keyboard 5 18 In 12 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com ER A ERE EE SERRA eR ERNE EN
119. ration is subject to the following two conditions 1 this device may not cause harmful interference and 2 this device must accept any interference received including interference that may cause undesired operation This device has been tested and found to comply with the EMC standards for emissions and susceptibility established by the European Union at time of manufacture This digital apparatus 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 RADIOELEC TRIQUES DEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES DE CLASS A PRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIO ELECTRIQUE EDICTE PAR LE MINISTERE DES COMMUNICATIONS DU CANADA FCC Warning Warning Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment Note This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radiofrequency energy and if not installed and used in accordance with the instruction manual may cause h
120. rmly 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 When tubes are cooled to below ambient temperatures a ring of condensa tion 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 complete 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 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 polyester backing allows easy inspection of sample wells Microseal B clear adhesive Seals are ideal for thermal cycling in all polypropylene and polystyrene microplates Microseal F aluminized foil acts as a barrier against evaporation from 20 C to 105 C In addition to cold storage applications it can also be used for thermal cycling sample volumes gt 25 yl 96 well or gt 5 ul 384 well The foil is thin enough to pierce with a pipet t
121. rogram The next step is the gradient step Highlight Step 2 in the program listing Select Gradient Gradient Step Step 3 Lower Temperature 1 E Higher Temperature E Hrs Min Sec Time CEL Forever Options Extend Time Set parameters Preview DK Cancel Entering a Gradient Step The Gradient Step window indicates the step number and includes fields for specifying the Lower Temperature and 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 1 C 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 Enter 45 C Select the Higher Temperature field Enter 65 C Select the Time Min field Enter 3 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 31 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual The newly created gradient can be previewed graphically e Select Preview Gradient Preview xl This distribution of temperatures specified by the gradient should be reviewed and any changes to the gradient limits made before a program run
122. rogram 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 4C but colder temperatures require considerably more power to maintain and are unnecessary in most circumstances in the opinion of Bio Rad scientists Adding a forever incubation step is identical to programming a temperature step with the exception of selecting the Forever box after specifying the incubation temperature The duration of the step will be displayed as a blue infinity symbol Program GRAPHIH DJ Lid Mode constant 100 00 Unused temperature step Step 5 forever Add Step after Control Mode carc tempertaure step Temp Grad Golo Forever Delete Step Save Save As Save Run 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 gra dient step column Our completed program appears as follows Program GRAPHH LJ Lid Mode constant 100 00 Add Step after LJ Control Mode carc Temp Grad Golo Iv Forever Delete Step Save i Save Run Cancel Select Save to save the completed program GRAPH 1 Note If your completed graphical program still bears the name TEM
123. rotected by copyright laws and international copyright treaties as well as other intellectual property laws and treaties The SOFTWARE PRODUCT and THIRD PARTY SOFT WARE are licensed to LICENSEE not sold 2 4 No License to Methods of Use No license is granted hereunder to any process method or use for which the SOFTWARE PRODUCT may be used either by itself or in combination with an analytical instrument except to the extent such a process is described in the MANUAL and BIO has the right to grant a license to such a process hereunder 3 SOFTWARE PRODUCT LIMITED WARRANTY 3 1 Limited Warranty BIO warrants that the SOFTWARE PRODUCT will perform sub stantially as described in the MANUAL if used in the manner described therein for a period of ninety 90 days from the date of receipt BIO also warrants that the media on which the software is distributed is free from defects in mate rials and workmanship To the extent allowed by applicable law implied warranties on the SOFTWARE PRODUCT if any are limited to the same ninety 90 days 3 2 Customer Remedies BIO s entire liability and your exclusive remedy shall be at BIO s sole option either a return of the purchase price paid by LICENSEE if any for the SOFTWARE PRODUCT that does not meet BIO s limited warranty or b repair of the SOFTWARE PRODUCT that does not meet BIO s limited warranty or c replace ment of the SOFTWARE PRODUCT that does not meet BIO s limited warranty pro
124. rst open the program GRAPH 1 in basic mode as described above e Select the gradient step e Select Delete Step e Select step 2 the temperature incubation at 92 C Recall that new steps are added AFTER the selected step e Select Temp 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 Click once to accept the change Our example program GRAPH 1 now appears as follows Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 6 5 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual Program GA PH41 LJ Lid Mode constant 100 00 Add Step after Control Mode carc Temp Grad Golo v Forever Delete Step Save Save As Save Hun Cancel Editing an Advanced Program Highlighting and Selecting Program Steps Several important factors determine step selection e The insertion point for new steps is AFTER the step highlighted in the pro gram listing This highlighting is accomplished with a SINGLE click e Double clicking on a program step will immediately open the appropriate step editing window for the highlighted step The windows for editing are the same as those for step creation e Selection can also be done via the left mouse bu
125. s Select Open Select Basic Mode gt The open program window will appear Select the folder FOLDER1 Select the program GRAPH 1 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 Gradient from 45 C to 65 C for 00 03 00 KR O Cycle to step 2 for 29 more times 5 Incubate at 10 C forever End Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Managing and Editing Programs W After running the above program analysis of the resulting sequencing data indicated 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 fi
126. s Permits remote control of Alpha unit lid opening available in 96 384 and flat block formats 2 4 Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com ao Layout and Specifications W DNA Engine Tetrad 2 Specifications Thermal range 0 105 C but no more than 30 C below ambient temperature 10 105 C for the Slide Chambers unit Temperature accuracy 0 3 C of programmed target at 90 C NIST traceable 0 4 C for dual Alpha units Temperature uniformity 0 4 C well to well within 30 seconds of arrival at 90 C 0 5 C for dual Alpha units Speed of ramping Up to 3 C sec for all single and dual block Alpha units Up to 1 2 C sec for the Slide Chambers Alpha unit Sample capacity Varies with installed Alpha unit Line voltage 200 240 VAC Frequency 50 60 Hz Power 1600 W maximum Fuses Two 6 3 A 250 V 5 x 20 mm Displays One 1 4 size VGA screen 320 x 240 16 colors Ports One 9 pin RS 232 serial port One ethernet port Program Capacity 1 000 typical Weight 21 6 kg base only Size 47 x 61 x 16 cm I x w x h base only Projected Life Expectancy 10 years of normal usage 2 protocol runs day 7 years of heavy usage consistently exceeding 2 protocol runs day Gradient Specifications 96 Well
127. s 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 2 ml tubes and 0 5 ml tubes 96 Well Polycarbonate Microplates These microplates present the largest risk of contamination Polycarbonate is somewhat permeable both to water and the S 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 2 ml 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 permeable to the 35S breakdown product Either way there have been reports of 35S passing through the walls of 0 2 ml tubes of several different brands during thermal cycling No data are yet available on radioactive contamination with polypropylene microplates 0 5 ml Polypropylene Tubes Contamination problems are rarer with this type of tube but instances have been reported Tech Support 1 800 4BIORAD e 1 8
128. s 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 Front end OK Are the cusor keys working Try navigating a window with them proceed Does the external mouse port function Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 10 9 E DNA Engine Tetrad 2 Thermal Cycler 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 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 communcating with 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 DNA Engine Tetrad 2 system check out OK we recommend positive controls for troubleshooting purposes
129. 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 There are three scenarios for programming this initial temperature step 1 If there are no steps displayed e Select lt Temp gt A 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 Ifthe first step displayed is a gradient step e Select the gradient step step 1 e Select Temp Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 21 HM DNA Engine Tetrad 2 Thermal Cycler Operations Manual 5 22 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 Delete Step 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 If the first step displayed is a temperature step or if a temperature step was added as indicated in scenarios 1 or 2 Position the cursor text pointer in the temperature field of step 1 and select by clicking once A flashin
130. sible The cycler is now ready to accept edit and execute programs Programs Utilities Command View Block is Inactive UserName GUEST Block 14 30 y 14 30 Temperature Time Block 28 C Step 00 00 00 en 228 C Remang 00 00 00 21 3 C Cycle us Instant Stop Pause m 4 2 Tech Support 1 800 4BIORAD 1 800 424 6723 www bio rad com Operation Using the Control Panel The control panel see figure 2 2 includes a display screen cursor keys a numeric keypad and enter key Display Screen e The display screen is a 1 4 size VGA screen for displaying thermal cycler condi tions and programs Operation Keys e Navigation keys left and right arrows next back Use to move around within the display screen e Numeric keypad Use to enter numeric values e Enter key Use to accept specific programming additions and modifications Note The external mouse is used to execute selected commands access menus and to toggle through selection options in a list Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 4 3 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual Block Status Lights e When illuminated these blue lights indicate whether the left and or right Alpha units are in use Using the Data Ports The DNA Engine Tetrad 2 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
131. t 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W The DNA Engine Tetrad cycler comes with several preprogrammed thermal cycling protocols These protocols are listed in Appendix C This chapter describes how to create your own protocols while the next chapter describes how to modify both the preprogrammed protocols and any you create yourself In this chapter we revisit the setup of the front panel specifically those items used in program input We describe the conventions used as well as the various programming steps and what they accomplish We make suggestions regarding the translation of a cycle sequencing protocol into a Tetrad 2 program Finally we use a cycle sequencing example to illustrate the pro gramming process step by step This manual documents version 4 00 of the DNA Engine Tetrad 2 front end software Front Panel Setup The various components of the DNA Engine Tetrad 2 cycler s control panel see figure 2 2 allow the operator to enter navigate and manipulate programs These programs are nec essary to control the various dynamic capabilities of the DNA Engine Tetrad 2 cycler Note Chapter 4 covers the basic operation of the DNA Engine Tetrad 2 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 10 cm x 12 5 cm located at the lef
132. t side of the control panel It displays all DNA Engine Tetrad 2 cycler operating parameters and can be controlled by the cursor buttons external mouse and the numeric keypad Navigation Keys There are four navigation 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 external mouse device The next back keys are primarily used to move through all available buttons or options in any given window The left right keys are used to navigate through menu bars Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com 5 3 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual 5 4 Enter Key This is located below the navigation keys and is used to accept specific program ming additions and modifications Note The external mouse is used to execute selected commands access menus and to toggle through selection options in a list Numeric Keypad This is located to the right of the navigation buttons and consists of a typical numeric keypad numbers 0 9 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 Programming Conventions Before starting the programming process let s review the conventions used here e lt lt gt gt will be used to indicate actual keys on
133. t temperature for long periods of time Modifying Block Control Programs for Calculated 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 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 DNA Engine Tetrad 2 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Creating Programs W Choosing a Lid Control Mode When a sample is heated condensation on the tube cap 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 DNA Engine Tetrad 2 cycler can control lid temper ature in three possible ways Constant Tracking or Off Constant Mode This mode maintains the inner
134. ten 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 1 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 Now that we ve made some important decisions regarding the implementation of our program we are ready to begin entering steps Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 13 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual Entering Program Steps From our example we are ready to enter a new program To execute a selected com mand left click the mouse Start up procedures for the DNA Engine Tetrad 2 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 DNA Engine Tetrad 2 cycler has completed its boot up sequence the Status window will be visible Block selection menu Programs Utilities Command View Block is Inactive Block 14 30 Se User Name GUEST Block status line Program display box Temperature Time Block 228 C Step 00 00 00 Sample 278 C Remaining 00 00 00 Lid 21 9 C Cycle 0 Instant Stop Pause Skip Block sample and lid temperatures are displayed for the convenience of the operator along with the c
135. 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 Programs in the Status window e Select Delete Folder from the drop down menu e To delete a folder highlight it and select lt Delete gt Delete Folder x You will be presented with a confirmation screen asking you to verify the deletion Select lt Yes gt to delete the folder Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 6 11 F4 Running Programs Using the Instant Incubation Feature lseleeeer ee 7 2 Running Programs 5x sis v y E UR EXOE A eee ees 7 4 The R n Program Window s bicis ee eredi we pem ac edes E 7 5 During the RUN sour ERE a GERE EN ERE REUS Cte 7 6 HUn StdluS neose nia iw edit e bee UR te Do editas en ere tede dus te HR eee 7 6 Terminating A AUN ova de isan ade e Dio ase at ate ee TI TIT 7 8 P sing R es ming a RUM sad 7 8 SKIDPING Step rca eg ACE a aa AS ARA AAA AAA 7 8 Inaccessible Features oooooooommmnrnn hh hh hn 7 8 Running Multiple Programs sess hh hh hh hh hh ht 7 9 ll DNA Engine Tetrad 2 Thermal Cycler Operations Manual In Chapter 5 we translated an experimental protocol into functional Tetrad 2 programs In Chapter 6 we edited program steps In this chapter we explore the impl
136. these ports Operating Alpha Units Note Operation of the Slide Chambers Alpha unit will not be discussed owing to the many differences between this type of Alpha unit and the others Please see the Slide Chambers Alpha Unit Operations Manual for operating instructions Note Moto Alpha units are installed and removed as described below See the Moto Alpha Unit Users Manual and The Moto Alpha Unit section in Chapter 5 for information on opening and closing Moto Alpha units Installing an Alpha Unit Caution Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2 cycler turned on electrical arcing can result Read the safety warning at the beginning of this manual on page iv regarding electrical safety when inserting or removing an Alpha unit 1 Turn off the DNA Engine Tetrad 2 cycler see the Caution above 2 Hold the Alpha unit at its front and back edges 3 Lower the Alpha unit into the DNA Engine Tetrad 2 base leaving at least 3 cm between the front edge of the Alpha unit and the center of the DNA Engine Tetrad 2 cycler base 4 Raise the handle at the back of the Alpha unit and slide the block toward the center of the base as far as it will go see figure 4 14 5 Push the handle down until it is completely vertical see figure 4 1B firm pres sure 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 Tetrad 2 cyc
137. through the remote command set The Moto Alpha has four options for Lid Pressure settings OX no pressure applied for microarrays or slides 0 5X for 8 or more tubes or strips 1 0X for any plate sealing options and 2 0X for custom applications Lid Angle The angle that the fully opened lid makes with the block is also set either through the front panel of the cycler or through the remote command set The Moto Alpha has four options for Lid Angle settings 70 80 90 or 100 open With the Tetrad 2 operation software the lid pressure and lid angle settings are set in the mode selection menu see below and are saved directly to individual protocols Please see the Moto Alpha Unit User s Manual for further instruction Set Power Lid Options Set Closing Force Set Opening Angle High High r 2 r 100 r 90 r 80 m 70 Low Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Managing and Editing Programs Opening a Program circa AAA RARA ADA AAA RE RN E RES 6 2 Opening a Program in Advanced Mode csse hem 6 2 Opening a Program in Basic Mode c 12 0 be detowe ew tiaeedsewaateudies ad 6 4 Editing a Program ei vie ria Sk eee a a ee ws ERR T deed 6 4 Editing a Graphical Progra uia dcr iio ba rem bird e e lo em e 6 5 Editing an Advanced Program sessi dle aptat OR TIER aae E IC RA dasa 6 6 Highlighting and Selecting Program Steps llssuslsus 6 6 Deleting a SIED M A AA ed ook 6 6 Inserting
138. ts Ensure that the area where the DNA Engine Tetrad 2 cycler is installed meets the following conditions for reasons of safety and performance e Nonexplosive environment e Normal air pressure altitude below 2000 m e Ambient temperature 5 31 C e Relative humidity of 10 9096 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 DNA Engine Tetrad 2 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 3 3 M DNA Engine Tetrad 2 Thermal Cycler Operations Manual 3 4 Power Supply Requirements The DNA Engine Tetrad 2 cycler requires 200 240 VAC 50 60 Hz and a grounded outlet on a minimum 20 A line The DNA Engine Tetrad 2 cycler can use voltage in the specified range without adjustment so there is no voltage setting switch Note Do not cut the supplied power cord and attach a different connector Use a one piece molded connector If required additional dedicated power cords may be purchased through Bio Rad Laboratories Air Supply Requirements The DNA Engine Tetrad 2 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
139. tton In order to insert or edit steps the user of the DNA Engine Tetrad 2 cycler should become familiar with these conventions By default in this manual we use the single mouse click to highlight a step and a double click to select it Deleting a Step The Delete Step 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 e Highlight step 3 by positioning the pointer over the first line of step three and clicking once e Select Delete Step The gradient step will be deleted and the following steps renumbered For example the goto step previously step 4 will become step 3 6 6 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com Managing and Editing Programs W Inserting a Step Now we will insert an annealing temperature step e Highlight step 2 Recall that new steps are inserted AFTER the highlighted step Select Temp 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 param eters 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
140. ull force and effect 7 3 Intellectual Property Rights All title and intellectual property rights including copyrights in and to the SOFTWARE PRODUCT the THIRD PARTY SOFTWARE the accompanying printed materials including the MANUAL and any copies of the SOFTWARE PRODUCT are owned by BIO or its suppliers All rights not expressly granted are reserved by BIO 7 4 U S Government Restricted Rights The SOFTWARE PRODUCT and any docu mentation are provided with RESTRICTED RIGHTS Use duplication or disclosure by the Government is subject to restrictions as set forth in subparagraph c 1 ii of the Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 or subparagraphs c 1 and 2 of the Commercial Computer Software Restricted Rights clause at 48 CFR 52 227 19 as applicable Manufacturer is Bio Rad Laboratories Incorporated 2000 Alfred Noble Drive Hercules CA 94547 7 5 Export Restrictions The SOFTWARE PRODUCT the THIRD PARTY SOFTWARE any part thereof and any accompanying documentation including the MANUAL shall not be exported or re exported outside of the United States without BIO s prior written permission and if BIO gives such permission the SOFTWARE PRODUCT THIRD PARTY SOFTWARE any part thereof or accompanying doc umentation shall not be exported or re exported to any country person entity or end user subject to U S export restrictions 7 6 Applicable Law This AGREEMENT shall be governed b
141. upply 3 4 In 1 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual ensuring air is cool enough 3 5 troubleshooting problems with 3 5 Alpha units closing 4 6 dual block models 2 4 installing 4 4 4 5 Moto Alpha unit 2 4 6 38 opening 4 6 removing 4 5 single block models 2 4 slide block 2 4 B 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 air vents 9 2 biohazardous materials 9 3 chassis and block 4 14 9 2 radioactive materials 4 14 9 3 solutions to use 4 14 9 2 Condensation in tubes following holds 4 8 Constant temperature incubator use as 7 2 Constant mode 5 11 Control panel 2 2 4 8 5 3 block status lights 4 4 cursor buttons 4 3 5 3 In 2 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com display screen 5 3 layout 2 2 numeric keypad 4 3 5 4 using 4 3 Conventions programming 5 4 Copying programs 6 9 Cycler log 7 7 D Deleting a folder 6 11 Deleting a step advanced program 6 6 Deleting a step graphical program 5 21 6 5 Deleting programs 6 10 Display screen location of 2 2 E Editing a program advanced 6 6 6 8 deleting a step 6 6 editing a step 6 7 inserting a step 6 7 selecting steps 6 6 graphical 6 5 6 6 deleting a step 5 21 6 5 editing a step 5 20 6 5 inserting a step 5 21 6 5 selecting steps 5 20 6 5 Environmental requirements 3 3 3 5
142. urrent cycle number The time remaining in the current step and in the program are also indicated At the screen bottom the program control buttons Run Instant Stop Pause Skip and Graphs 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 Graphs 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 Status button By leaving your cursor in the same position in the window and clicking the external mouse 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 selected The Program Display box will list steps for the program currently running on the selected block 5 14 Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com Creating Programs W The menu bar at the top of the Status window includes four submenus lt Programs gt lt Utilities gt lt Command gt and lt View gt These submenus provide the operator with paths for maneuvering through the various DNA Engine Tetrad 2 software windows For the purposes of this chapter we will be primarily concerned with the Progr
143. vided that under a b or c above the SOFTWARE PRODUCT that does not meet BIO s limited warranty is returned in its entirety to BIO or the authorized seller from which the SOFTWARE PRODUCT was purchased complete with a dated proof of payment within 90 days of the date of delivery Any repaired or replaced SOFT WARE PRODUCT will be warranted as described in Paragraph 3 1 for the remainder of the original warranty period or thirty 30 days whichever is longer 3 3 Warranty Void The limited warranty of Paragraph 3 1 and remedies of Paragraph 3 2 are void if a failure of the SOFTWARE PRODUCT has resulted from accident abuse or misapplication of the CYCLER or SOFTWARE PRODUCT b the SOFTWARE PRODUCT is used with any operating system other than operating system included in the CYCLER c the SOFTWARE PRODUCT is used for the analysis of data generated by any analytical instrument other than the BIO analytical instruments with which the SOFTWARE PRODUCT was designed to be employed and which are specifically listed in the MANUAL Tech Support 1 800 4BIORAD 1 800 424 6723 e www bio rad com B 3 M DNA Engine Tetrad 2 Thermal Cycler Operations Manual B 4 d failure of the SOFTWARE PRODUCT has resulted from anyone other than BIO or its authorized representative installing or running any software on the computer running the SOFTWARE PRODUCT other than the SOFTWARE PRODUCT itself or e for failure to comply with any of the pro
144. visions of this License Agreement 3 4 NO OTHER WARRANTIES TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE LAW BIO DISCLAIMS ALL OTHER WARRANTIES AND CONDITIONS EITHER EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO IMPLIED WAR RANTIES OR CONDITIONS OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE TITLE AND NON INFRINGEMENT WITH REGARD TO THE SOFTWARE PRODUCT 4 0 LIMITATION OF LIABILITY 4 1 LIMITATION OF LIABILITY TO THE MAXIMUM EXTENT PERMITTED BY APPLIC ABLE LAW IN NO EVENT SHALL BIO OR ITS SUPPLIERS OR EMPLOYEES BE LIABLE FOR ANY SPECIAL INCIDENTAL INDIRECT OR CONSEQUENTIAL DAM AGES WHATSOEVER INCLUDING WITHOUT LIMITATION DAMAGES FOR LOSS OF BUSINESS PROFITS BUSINESS INTERRUPTION LOSS OF BUSINESS INFORMATION OR ANY OTHER PECUNIARY LOSS ARISING OUT OF THE USE OF OR INABILITY TO USE THE CYCLER OR SOFTWARE PRODUCT OR THE FAILURE TO PROVIDE SUPPORT SERVICES EVEN IF BIO HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES IN ANY CASE BIO s ENTIRE LIA BILITY UNDER ANY PROVISION OF THIS AGREEMENT SHALL BE LIMITED TO THE PURCHASE PRICE PAID BY LICENSEE FOR THE CYCLER AND SOFTWARE PRODUCT 4 2 The CYCLER SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are not intended for HIGH RISK ACTIVITIES as defined below In particular the CYCLER SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are not fault tolerant and are not designed manufactured or intended for use or resale as on line control equipment in hazardous environments requir
145. y brands provide a good and consistent fit Bio Rad thin walled 0 5 ml tubes are designed for precise block fit and tight sealing of reactions down to 10 yl Thin Walled vs Thick Walled Tubes The thickness of sample tubes directly affects the speed 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 2 ml Tubes All types of thin walled 0 2 ml tubes may be used Bio Rad offers high quality 0 2 ml 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 10 yl Several varieties of microplates are available from Bio Ra
146. y the laws of the State of California 7 7 Survival Sections 2 2 2 3 2 4 3 4 5 and 7 shall survive termination cancellation or completion of this AGREEMENT Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com B 5 2 step Fast PCR protocol using iTaq polymerase iTAQ FST Step Temp Time 1 98 30 2 92 01 3 70 10 4 GOTO 2 34X 5 72 115 6 END 2 step PCR protocol using iProof high fidelity polymerase for small intermediate and long templates iPRF1kb 1 95 2 00 2 95 05 3 72 30 4 GOTO 2 219X 5 72 10 00 6 END iPRF8kb 95 2 00 2 95 05 3 72 4 00 4 GOTO 2 19X 5 72 10 00 6 END iPRF15kb 1 95 2 00 2 95 05 3 72 7 30 4 GOTO 2 19X 5 72 10 00 6 END Appendix C Factory Installed Protocols Standard 2 step PCR protocol STD 2 Step Temp Time 1 95 4 00 2 95 30 3 65 30 4 GOTO 2 29X 5 4 6 END Standard 3 step PCR protocol STD 3 1 95 4 00 2 95 30 3 55 30 4 72 30 5 GOTO 2 29X 6 72 7 00 7 4 8 END Touchdown PCR protocol TCHDOWN 1 95 4 00 2 95 30 3 60 30 Increment 0 5 C cycle 4 72 30 5 GOTO 2 29X 6 95 30 7 45 30 8 72 30 9 GOTO 6 29X 10 72 7 00 11 4 12 END C 1 M DNA Engine Tetrad 2 Thermal Cycler Operations Manual 2 step RT PCR protocol RTPCR 2 Step Temp Time 1 37 60 00 2 95 5 00 3 95 30 4 65 30 5 GOTO 3 39X 6 72 7 00 7 4 8 END 3 step RT PCR protocol RTPCR 3 1 37 60 00 2 95 5 00 3 95 30 4 55 30
147. yme 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 Enter 60 C in the Temperature field Enter 1 in the Time Min field Select Extend Time Select Set Parameters for the Extend Time option The Extend Time window appears Extend Time x M Increase Decrease by Seconds per Cycle OK Cancel Select Increase Position the cursor over the by __ seconds per cycle field and select 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 Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 33 E DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 GoTo Step Step 4
148. zed lid it may be desirable to include steps in the Tetrad 2 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 For further instruction on running the Moto Alpha unit see The Moto Alpha Unit which appears at the end of this chapter 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 sec tion 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 Temperature enter 60 as an example and Time enter 30 seconds as an example e Select Slow Ramp Select Set Parameters for the Slow Ramp option The Slow Ramp window appears Slow Ramp Option Ea Ramp to 60 0 C C per second OK 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 follows Incubate at 60 0 C for 00 00 30 Ramp to 60 C at 0 5 C per second Tech Support 1 800 4BIORAD e 1 800 424 6723 e www bio rad com 5 35 B DNA Engine Tetrad 2 Thermal Cycler Operations Manual

DNA Engine Tetrad® 2 - American Instrument Exchange, Inc.

Contents

Download Pdf Manuals

Related Search

Related Contents

DNA Engine Tetrad&reg; 2 - American Instrument Exchange, Inc.

Contents

Download Pdf Manuals

Related Search

Related Contents

DNA Engine Tetrad® 2 - American Instrument Exchange, Inc.