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1. 6 10 Programming an indefinite Hold 6 10 Creating a program that requires manual stepping 6 6 10 Editing While Entering a Program 6 10 Changing the last value entered or last menu option chosen sse 6 10 Changing all the values in the step being entered eee petet 6 11 Changing values in earlier steps of program being entered 6 11 Programming Deleting an Incomplete Program 6 12 Deleting a Program fromemory 6 12 Listing a Program 6 12 Printing a 2 55 5 6 13 6 1 Bl MiniCycler Operations Manual MiniCycler programs consist of a series of steps encoding a protocol These steps are run using one of two temperature control methods block con trol or probe control Programs may contain six types of steps Two of the step types are man datory and four are optional 1 Temperature step mandatory Sets a temperature for the block and the length of time it is held at that temperature The MiniCycler brings the block to this temperature at its maximum rate of heating or cooling up to 2 4 C sec unless modifying instructions are added to the program 2 GoTo step optional Causes the program to cycle back to an earlier
2. 0150 Operations Manual Version 4 0 MJ RESEARCH Inc Waltham MA Copyright 1999 Research Inc rights reserved PTC 150 MiniCycler Hot Bonnet Chill out and Microseal are trademarks of MJ Research Inc 06108 05 Thermal cyclers can be used for a number of purposes including the polymerase chain reaction PCR PCR is covered by patents owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd who have granted exclusive and nonexclusive licenses for some types of applications Roche and its licensees provide end user licenses within their respective fields These licenses have dif ferent terms depending on the particular application of PCR and different rules may apply in different countries Anyone who intends to use MJ Research equipment to do PCR is encouraged to contact Hoffmann La Roche for more information at one of the addresses below In the United States In other nations Kathe Kiehn Dr Agnieszka Junosza Jankowski Licensing Manager PCR Licensing Manager Roche Molecular Systems Inc F Hoffmann La Roche Ltd 1145 Atlantic Avenue Building 222 350 Alameda CA 94501 USA CH 4002 Basel Switzerland 510 814 2970 41 61 687 3031 Fax 510 814 2977 Fax 41 61 687 2113 Contents POCO aoo Conventions e ueni adv Pace tud EE d 10 FEM 1 1 2 bayou
3. 4 7 Loading sample vessels 4 8 Loading tubes and 4 8 Using the sponge rubber pad to improve tube contact with the block 4 9 Using oil to improve thermal contact 4 9 Using the Probe 55 4 9 Customizing the probe vessel 4 10 e 4 10 Loading and connecting the probe 4 11 Running a protocol under probe control 4 11 Tube Microplate and Sealing System Selection Chart Safety Warning Regarding Use of S Nucle otides 4 1 MiniCycler Operations Manual Turning the MiniCycler On Move the power switch to 1 the On position A self test of the heat pump will usually begin running see below Its progress is tracked in screen in the LCD window SELF TEST This screen disappears within 10 seconds If a problem is detected an error message will be displayed Note If either the heat sink or the block is not between 17 C and 30 C the machine will skip the self test If the self test does not detect any problems the Run Enter Menu is dis played RUN Enter Program Program Using the Control Panel 4 2 The control panel see fig 2 3 includes operation keys an LCD window for displaying programming and machine status text and a nu
4. 4 Warning MiniCycler incorporates neutral fusing which means that live power may still be available inside the unit even when a fuse has blown or been removed Unplug the MiniCycler from the electrical outlet before opening its case Never connect a power cord to the machine when the case is Open Figure 8 1 Changine a fuse 8 3 Bl MiniCycler Operations Manual Follow this procedure to change the fuse 1 Move the power switch to the 0 off position Disconnect the power cord from the side of the machine 2 Insert one corner of a small flat head screwdriver into the slot at the left side of the fuse block fig 8 1A Gently pry the block loose and pull it down and out The fuses will be visible fig 8 1B 3 Remove the fuses and examine them A flat head screwdriver may be used to pry the fuses loose if necessary A broken central wire or dark ened glass indicates that a fuse has blown and should be replaced 4 Gently press the fuse block back in place and reconnect the power cord 8 4 Troubleshooting Error Messages 9 2 Problems Related to Protocols 9 3 Problems Related to Machine Performance 44 64 9 5 9 1 Bl MiniCycler Operations Manual Error Messages Error Message Check ADC Check Block Sensor Check EEPROM Heat Pump Not Functioning HS Overheating Check Air Flow 9 2
5. control panel 2 3 front view 2 2 M Microseal adapter 4 7 P Packing checklist 3 2 Power outage resuming a protocol after 5 5 Power supply requirements 3 3 Probe adding oil to 4 12 calculating amount table 4 12 connecting to block 4 12 correctly installed 4 11 customizing vessel 4 11 function of 4 10 running a protocol with 4 12 thermistor 4 10 Probe 2 5 Programming designing a program 6 2 translating a protocol into a program 6 2 using the GoTo step 6 3 editing changing last value or option entered 6 10 changing values a step 6 11 deleting a program from memory 6 12 deleting an incomplete program 6 12 initiating 7 2 slope steps 7 4 temperatures and cycling values 7 2 time values 7 3 entering program steps and options 6 5 end step 6 10 Extend option 6 8 GoTo step 6 6 Increment option 6 7 indefinite hold how to program 6 0 manual stepping how to program 6 10 slope step 6 9 temperature step 6 5 initiating a program 6 4 listing a program 6 12 naming a program 6 5 printing 6 13 types of programming steps 6 2 Protocols Instant Incubation 5 6 manually stepping through 5 4 pausing while running 5 5 problems with 9 3 resuming after power outage 5 5 running custom 5 2 protocol completion screen reading 5 4 runtime screen reading 5 3 under Probe Control 5 3 with Hot Bonnet 5 2 stopping while running 5 5 S Safety general instructions 1 2 warnings and cautions 4 15 Sample vessels and Microseal a
6. using empty tubes if necessary Figure 4 1 Mounting the Hot Bonnet A Rear view of MiniCycler showing correct cable orientation for the Hot Bonnet B Hot Bonnet side view showing mounting lever C Correctly mounted Hot Bonnet i 25 Mounting pin 4 3 Bl MiniCycler Operations Manual 3 Plug the Hot Bonnet into its port at the back of the machine so that the cable is to the left of the connector as you face the back of the machine fig 4 1A 4 Raise the Hot Bonnet lever and set the lid into the block with the lever pointing to the right as you face the machine s front 5 Lower the lever as far as possible which locks the Hot Bonnet into place and lowers the heated lid onto the loaded tubes Figure 4 1C shows a correctly mounted Hot Bonnet Note Intermittent beeping is an indication that the heated lid is not being used correctly If a PTC 150 is beeping it is likely there are not four tubes of the appro priate height in each of the four corners of the sample block Placing a tube in each of the four corners of the sample block will allow the lid to seat squarely atop the block and the beeping will stop The Minicycler heated lid is unadjustable and designed to fit over standard sized flat capped 0 5 mL tubes on the 16 well cycler or standard sized 0 2 mL dome capped tubes on the 25 well cycler If you would like to use a non standard tube height or flat capp
7. Cause Analog digital converter not giving proper output Block s temperature sensor or sensor circuitry is malfunctioning User Memory failing to store data Heat pump is not working so machine has shut down Machine is not getting enough air or air being taken in is not cool enough Action Contact MJ or your distributor Block may need servicing Contact RESEARCH or your distributor Turn machine off then on again If error message appears again contact MJ Research or your distributor Cycler needs servicing Contact MJ RESEARCH or your local distributor Ensure machine is at least 10cm away from walls and other equipment Make sure air being taken in is cool see chap 3 If message persists cycler may need servicing contact RESEARCH or your distributor Problems Related to Protocols Problem Cause Troubleshooting Action Reaction is working but broad low mole cular weight band is seen in gels Reaction working but unexpected extra products or smear is seen Primer dimer material often produces a broad band in the lt 100bp region of gels Nonspecific hybridization occur ring during setup Reaction component concentration too high or too low Annealing temperature too low Template not of sufficient purity Multiple templates or host DNA in sequencing reactions Continued If obtaining appropriate reac tion product s no ne
8. RESEARCH Inc is not responsible for special incidental or consequential damages resulting from any breach of warranty or under any other legal theory including downtime lost samples or experiments lost reagents lost profits goodwill damage to or replacement of equipment prop erty and any costs of recovering or reproducing experimental results and data Exclusions This warranty applies only to machines sold in the U S A Under no circumstance will MJ RESEARCH ship a repaired or replaced machine or grant a refund of purchase price to a user in a nation which there was an authorized MJ distributor at the time of purchase THIS WARRANTY IS NOT TRANSFERABLE FROM THE ORIGINAL PURCHASER TO A SUBSEQUENT OWNER FUR THERMORE THIS WARRANTY DOES NOT APPLY TO INSTRUMENTS USED OUTSIDE THE U S A EXCEPT WHEN EXPRESSLY AUTHORIZED IN WRITING BY MJ RESEARCH INC U S Domestic Extended Warranty Optional MJ RESEARCH Inc will offer to each ORIGINAL PURCHASER of an MJ BRAND thermal cycler the opportunity to purchase an extension of the warranty coverage explained above for an additional two years The coverage must be purchased through a purchase order received by MJ RESEARCH Inc within 30 days of receipt of the offer of extended warranty or the offer to renew the extended warranty These offers apply only to machines sold and used in the U S A B 1 Operations Manual Some states
9. Run Enter Menu is displayed Ascreen allowing entry of the incubation temperature will be displayed TEMP Type any incubation temperature from 9 0 to 105 0 then press lt Pro ceed The MiniCycler will incubate the sample at the specified tempera ture When the sample block reaches the incubation temperature a timer will begin running in the lower right hand corner of the screen To stop and start the timer press Pause To stop an instant incubation press Can cel or Stop see Stopping a Running Protocol Designing New 6 2 Translating a Protocol into a program 6 2 Using the GoTo Step to write short programs 6 2 Choosing a temperature control method 6 3 Block control eee 6 3 Probe control essen 6 3 Entering new program 6 4 Initiating the program eere 6 4 Naming the program eere 6 5 Entering the program 6 5 Entering a temperature step 6 5 Entering GoTo step 6 6 Entering an increment step 6 7 Entering an extend 6 8 Entering a slope step 6 9 Entering the End
10. be specified The desired temperature change for the step This is the difference between the temperature of the block at the beginning of the slope step and the desired block temperature at the end of the slope step For example if the final temperature of the preceding step is 92 C Programming and the desired final temperature of the slope step is 65 C the de sired temperature change is 27 C e The slope rate for the step This is a ratio of degrees per unit of time usually degrees per second Use the smallest convenient time incre ment to achieve the smoothest temperature curve for the step For example 0 1 C per second will give a smoother curve than 1 C per 10 seconds To enter a slope step select Option from the Enter Menu and press Pro ceed The Options Menu see above will be displayed Select Slope and press Proceed A screen allowing entry of the magnitude of the tem perature change for the step will be displayed Step 4 Slope _ Type anumber for the desired temperature change and press Proceed The number will be accepted and a screen allowing entry of the rate of heating or cooling will be displayed per Hrs Min Sec Type a number from 0 1 to 1 5 the maximum programmable tempera ture range for the numerator of the slope rate and press Proceed The temperature will be accepted and the cursor will move to Hrs Type a time value for the denominator of the slope rate A numb
11. block can become hot enough during the course of normal eration to cause burns or cause liquids to boil explosively Wear safety goggles or other eye protection at all times during operation Safe Use Guidelines The MiniCycler is designed to be safe to operate under the following conditions Indoor use e Ambient temperature 4 327 Relative humidity 10 90 noncondensing Transient overvoltage per Installation Category II IEC 664 Pollution degree 2 in accordance with IEC 664 Note operation at altitudes significantly above sea level may affect operation particularly heat dissipation 1 MiniCycler Operations Manual Electromagnetic Interference The MiniCycler has been tested and found to comply with the limits for a Class A digital device pursuant to part 15 of the US FCC Rules These limits are designed to provide a reasonable protec tion against harmful interference when the equipment is operated in a commercial environment This machine generates uses and can radiate radiofrequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio commu nications Operation of this machine in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense In addition the MiniCycler design has been tested and found to comply with the EMC standards for emissions an
12. box containing two lines of text Example Enter Program Program A programming option is termed selected when the cursor is positioned in front of it Use the Select keys see fig 2 3 to move the cursor In some screens selected items are also displayed in all capital letters Introduction Meet the 150 1 2 How to Use This 1 2 Important Safety Information 1 2 Bl MiniCycler Operations Manual Meet the PTC 150 MiniCycler Thank you for purchasing MJ PTC 150 MiniCycler Designed by a team of molecular biologists and engineers the MiniCycler will meet your needs for an easy to use reliable and compact programmable ther mal cycler e Easy to read programming interface for quick and painless program ming e Choice of block or probe control e Space saving design for easy setup and transportation e Instant Incubate feature for continuous temperature incubations e Customizable factory installed protocols e Hot Bonnet heated lid for oil free cycling How to Use This Manual This manual contains all the information you need to operate your MiniCycler safely and productively e Chapter 2 describes the physical characteristics of the MiniCycler e Chapters 3 5 describe the basics of installing and operating the MiniCycler e Chapters 6 and 7 describe programming the MiniCycle
13. cycle back to 1 is used in the example below Step 2 Go to step 1 Press Proceed The number will be accepted and a screen allowing Programming entry of an additional number of cycles will be displayed Step 2 Go to 1 more times Type the additional number of times the program should cycle back to the step 24 is used in the example below Step 2 Go to 1 24 more times Press Proceed The number will be accepted and the Enter Menu will be displayed again Use the Enter Menu to add another step to the program Entering an increment step To enter an increment step select Option from the Enter Menu and press Proceed The Options Menu will be displayed Step _EXTEND Increment Slope Select Increment and press Proceed A screen allowing entry of the ini tial incubation temperature for the step will be displayed Step 3 Temperature _ Type a temperature and press Proceed The number will be accepted and a screen allowing entry of an incubation time will be displayed Step 3 Min _ Sec Type anumber for Min and Sec Press Proceed to automatically enter a value of 0 6 7 Bl MiniCycler Operations Manual 6 8 When the numbers have been typed press Proceed The numbers will be accepted and a screen allowing entry of a temperature increment or decrement will be displayed Step 3 Add _ cycle Type a temperature from 6 0 to 6 0 C the maximum programmable range as
14. step for a specified number of times up to 9 999 times 3 Increment step optional Allows a progressive increase or decrease of temperature 6 0 to 6 0 C cycle each time a step is executed in a GoTo cycle useful in touchdown programs 4 Extend step optional Allows a progressive lengthening or shorten ing of a temperature step hold by 1 60 sec cycle each time a step is executed in a cycle useful for accommodating an enzyme with di minishing activity 5 Slope step optional Allow a slower than maximum rate of heat ing or cooling 6 End step mandatory Instructs the MiniCycler to shut down its heat pump because the last line of the program has run Designing a New Program 6 2 Using the GoTo step to write short programs The GoTo step allows programs of many repetitious steps to be short ened to just a few lines When the program encounters a GoTo step it returns to the specified step repeats that step and repeats all steps that follow back to the GoTo step When the program has returned or cycled back to the step the specified number of times the program moves on to the step that follows the GoTo step For example consider a basic cycle sequencing protocol consisting of 30 repeats of the denaturation and annealing extension steps Rather than listing all 60 steps use a GoTo step to design a short easy to enter pro gram Programming Raw program Shortened program 1 92 fo
15. the number of degrees by which the incubation temperature will increase or decrease each time the step is executed in a cycle Use a negative number to program a progressive decrease of temperature and a positive number to program a progressive increase When the number has been typed press Proceed The number will be accepted and the Enter Menu will be displayed again Use the Enter Menu to add another step to the program Entering an extend step To enter an extend step select Option from the Enter Menu and press Proceed The Options Menu see above will be displayed Select Ex tend and press Proceed Type the initial incubation temperature and incubation time as for an increment step see above and press Pro ceed The temperature and time will be accepted and a screen allowing entry of a time increment or decrement will be displayed Step 4 Extend S cyc Type a number from 60 to 60 the maximum programmable range as the number of seconds by which the incubation time will increase or de crease each time the step is executed in a cycle Use a negative number to program a progressive decrease of incubation time and a positive num ber to program a progressive increase When the number has been typed press Proceed The number will be accepted and the Enter Menu will be displayed again Use the Enter Menu to add another step to the program Entering a slope step To enter a slope step two values must
16. the pad to the metal plate under the lid When the lid is closed the pad will firmly seat tubes or microplates Using the Probe 4 8 An in sample temperature probe is available for MiniCyclers with the 16 or 25 well block The probe consists of a precision thermistor mounted in a thin walled plastic tube see fig 2 5 A thin wire encased in a small plastic tube runs from the thermistor to the probe s plug which is in serted into a jack at the side of the block A specific amount of oil is added to the probe tube see Adding the oil below to serve as the represen tative sample The tube is loaded into the block where it can serve as the control reference for any programmed target temperature between 0 and 100 C When a probe control protocol is run the MiniCycler controls the block s temperature to keep the probe at the programmed temperature using feedback information from the thermistor Protocols must be tailored to fit this control mechanism see chapter 6 Customizing the probe vessel For the most precise control of sample temperatures install the probe s wire and thermistor in the same type of tube that the samples will be placed in Follow these steps to customize the probe vessel 1 Cut the hinge to the probe tube s lid Gently remove the thermistor from the probe tube the lid will come off with it Operation 2 Remove the lid from the new probe tube Add the amount of oil speficied under Add
17. tubes in the PTC 150 MiniCycler KA 17 sec A difference E 27 M if 7 Block j Thin wall TN 1 VN f Thick wall ag ue E 15 sec difference 4 5 Bl MiniCycler Operations Manual Thin vs thick walled tubes The thickness of sample tubes directly affects the speed of sample heat ing and thus the amount of time required for incubations see fig 4 2 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 ramp 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 ramp rates so the faster heat transfer provided by thin walled tubes allows protocols to be significantly shortened For example in the reaction illustrated in figure 4 2 over 30 seconds can be saved per cycle by using thin walled tubes for an overall savings of 15 minutes in a 30 cycle run Sealing Sample Vessels 4 6 Water can evaporate from reaction mixtures during thermal cycling changing the concentration of reagents A layer of oil or wax will com pletely prevent evaporation from tubes and microplates but Microseal film mats or caps in conjunction with the Hot Bonnet
18. 10 In the example above the last entered step is temperature step The step number is 3 and the temperature for the step is 94 0 C The incuba tion time is displayed in the form of 00 00 00 The first two digits repre sent hours the second two digits represent minutes and the last two digits represent seconds The step in the example is programmed for a 10 second incubation At this point you may press the arrow keys to scroll through the program s steps To change temperature time and cycling values see chapter 7 Note You cannot change the program name step number or step type To change the program s name or the order or type of its steps delete the program see below and then re enter it After you have made all necessary changes press the right Select key until the Enter menu is displayed for the next step in the program Con tinue entering the new program Deleting an Incomplete Program To delete a program that you are in the process of entering enter the End step and press Proceed which will store the program Then delete the program from memory see below 6 11 MiniCycler Operations Manual Deleting a Program from Memory Any program in memory may be deleted To delete a program select Enter Program from the Run Enter Menu and press Proceed The Main Menu will be displayed Select Delete and press Proceed The deletion screen will be displayed for the first program in memory
19. Delete CUSTOM1 Press the Select keys until the name of the program to be deleted is displayed When the correct name is displayed press Proceed The program will be deleted and the Run Enter Menu will be displayed Listing a Program 6 12 Use the List option on the Main Menu to display all of a program s steps in the LCD window To enter List mode select Enter Program from the Run Enter Menu and press Proceed The Main Menu will be displayed Select List and press Proceed Press the Select keys until the name of the program to be listed is dis played then press Proceed The first step in the program will be dis played in the LCD window Press the right Select key to scroll forward and the left Select key to scroll backward through the list of steps To exit List mode scroll to the last step of the program then press the right Select key once The Run Enter Menu will be displayed Note No program values can be changed in List mode To change pro gram values see chapter 7 Programming Printing a Program If your MiniCycler has the optional 8 bit 25 pin parallel printer port it can print all the programs stored in the machine To do this follow the instructions under Listing a Program to enter List mode If a printer is connected to the printer port the list program screen will give you the option to print as well as list programs in the LCD window Select Print Prog
20. Heated lid are also effective and far less messy and inconvenient Sealing with oil or wax Mineral oil silicone oil paraffin wax or Chill out liquid wax may be used to seal tubes or plate wells Use only a small amount of oil or wax 1 3 drops 15 50 1 are usually sufficient Use the same amount of oil or wax in all sample vessels to ensure a uniform thermal profile Some paraffin waxes solidify at room temperature The wax can then be pierced with a micropipette and the samples drawn off Silicone oil and mineral oil can be poured off or aspirated from tubes if the samples are first frozen 15 to 20 C The samples are usually pure enough for analy sis without an extraction Chill out liquid wax available from MJ RESEARCH is an easy to use alternative to oil This purified paraffinic oil solidifies at 14 and is liq uid at room temperature By programming a hold step at low tempera ture the wax can be solidified at the end of a run A pipette can then be used to pierce the wax in the tubes and remove the samples The wax is dyed red to assist in monitoring its use clear Chill out wax is also avail able The dye has no adverse effects on fluorescent gel analysis of reac tion products Operation Sealing with the Hot Bonnet and caps or film sheets The inner lid of the Hot Bonnet heats to 108 C during a 99 second pre heat When heated the lid maintains a higher temperature in the upper part of the reaction
21. Note All tubes and Multiplate microplates are made from polypropylene plastic Microseal sealing film and Microseal mats are supplied in a 96 well format and must be cut to fit the Minicycler 4 11 Bl MiniCycler Operations Manual Safety Warning Regarding Use of 255 Nucleotides Some researchers have experienced a problem with radioactive contamination when using S in thermal cyclers This problem has occurred with all types of reaction vessels The Problem When 25 nucleotides are thermally cycled a volatile chemical breakdown product forms prob ably 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 Polycarbonate microplates These microplates present the largest risk of contamination Polycarbonate is somewhat perme able both to water and 55 breakdown product This problem is exacerbated when polycarbon ate plates are held at high temperatures for long periods of time or when the plates are sealed for oil free thermal cycling 0 2mL Polypropylene tubes and 25 well polypropylene microplates These tubes are manufactured with very thin walls to enhance thermal transfer The thin walls are somewhat fragile and can develop small cracks when subject to mechanical stress Undamaged thin polypropylene tubes may also be somewhat permeable to the S bre
22. OF THE U S A do not allow the exclusion or limitation of incidental consequential damages or limitations on how long an implied warranty lasts so the above limitation or exclu sion may not apply to you This warranty gives you specific legal rights You may also have other rights depending upon your state of residence All provisions of this warranty are voided if the product is resold repaired or modified by any one other than MJ RESEARCH or an authorized distributor B 2 Appendix Shipping Instructions for US Residents Users residing in the United States should follow these instructions for shipping a machine to MJ RESEARCH for factory repair or an upgrade Users outside of the United States should send ma chines to their distributor in accordance with shipping instructions obtained from the distributor 1 Call MJ Reszarcu to obtain a return materials authorization RMA number Machines returned without an RMA number will be refused by the Receiving Department 2 Thoroughly clean the machine removing oil and radioactive and other biohazardous sub stances To protect the health of our employees MJ RESEARCH will not repair or upgrade any machine that is excessively oily or that emits ionizing radiation upon arrival at our factory PLEASE ELIMINATE ALL BIOHAZARDS 3 Pack the machine in its original packaging If this has been misplaced or discarded call MJ RESEARCH to request shipment of pac
23. akdown product Either way there have been reports of 255 passing through the walls of microplates and 0 2 mL tubes of several different brands during thermal cycling 0 5 mL Polypropylene tubes Contamination problems are rarer with this type of tube but instances have been reported The Solution 1 Substitute the low energy beta emitter in cycle sequencing nucleotides are not subject to the same kind of chemical breakdown as 55 nucleotides and they have not been associated with volatile breakdown products 2 If 5 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 295 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 4 12 Operation 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 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 avail able in supermarkets Caution Harsh cleaning agents are corrosive to aluminum and must never be used o
24. alphabet selected Greek letters punctuation marks numbers To access the Japanese Katakana syllabary press the key A second press of returns the machine to Western characters When the character needed is displayed next to Name press Proceed Programming The character will be accepted and the cursor will move one space to the right Numbers and dashes may also be inserted by pressing the corre sponding keys on the keypad When the name is complete press Proceed once to accept the last char acter and again to accept the whole name If the name is already in use for a program a screen saying Name In Use will be displayed If this happens press Proceed then enter a different name Entering the program steps When the name has been entered CUSTOM1 is used in the following examples the Enter Menu will be displayed Step 1 _TEMP Goto Option End Use this menu to enter each step of the program Temp enters a temperature step GoTo enters a GoTo step Option enters an increment extend or slope step e End enters the End step Entering a temperature step To enter a temperature step select Temp from the Enter Menu then press Proceed The first Temp screen will be displayed Step 1 Temperature _ The first line of this screen shows the number of the step being pro grammed 1 is used in the example above The second line of the screen allows a target temperature in d
25. ample Probe Control _NO Yes Select Yes and press Proceed The protocol will begin running under probe control 49 Bl MiniCycler Operations Manual Figure 4 3 Correctly installed probe Table 4 1 Calculating the correct amount of oil Type of oil Heavy mineral oil Light mineral oil Silicone oil Silicone oil 4 10 Sigma number 400 5 M5904 DMPS 5X DMPS V Amount to use 1 x volume of buffer in individual sample tube 1 x volume of oil overlay 1 4 x volume of buffer in individual sample tube 1 x volume of oil overlay 1 7 x volume of buffer in individual sample tube 1 x volume of oil overlay 2 7 x volume of buffer in individual sample tube 1 x volume of oil overlay Operation Tube Microplate Sealing System Selection Chart Key Reaction vessel or sealer fits block without modification Reaction vessel or sealer must be cut to fit MJ Researcu Reaction Vessels Sealing Options for Oil Free Cycling 6 25 Description MJ RESEARCH Microseal Microseal 8 Strip 12 Strip 0 5ml 0 2 film M mat caps caps Catalog MSA 5001 MSM 1001 TCS 0801 TCS 1201 0 5ml tubes w caps thick wall TBI 0601 je peres ionic one je pem qmm p p neum p ram e FE tei mos p p tei mos p FB tini mos e
26. conforms to the applicable Directives and Standards of the European Union EU as described above Test data to verify this conformity are available for inspection at the MJ RESEARCH INc at the address listed below The instrument in question carries the CE mark on the nameplate MANUFACTURER S OFFICIAL pf Robert A lovanni Senior Electrical Engineer RESEARCH INC 590 Lincoln Street Waltham Massachusetts 02451 UNITED STATES OF AMERICA Tel 617 923 8000 Fax 617 923 8080 Date 8 5 99 E 1 Appendix Appendix F Factory Installed Protocols Note A programmed time of 0 00 00 means hold at this temperature forever Name ICEBUKET LIGATION 37 1 HR 37 6 HR 65 DENATURE BOIL CUT amp KILL Temp Control Method Block Block Block Block Block Block Block Block Step Temp amp Time 4 0 00 00 END 15 0 00 00 END 37 1 00 00 4 0 00 00 END 37 6 00 00 4 0 00 00 END 65 0 00 00 END 95 0 05 00 END 100 0 05 00 END 37 1 00 00 70 0 05 00 END Index A Air supply requirements 3 3 C Cleaning and radioactive materials 4 16 chassis and block 4 16 solutions to use 4 16 Control panel 2 3 keys 4 2 Documentation conventions iv E Environmental requirements 3 2 Error messages 9 2 H Hot Bonnet 2 4 mounting 4 3 I Instant Incubation 5 6 L Layout 150 back view 2 2
27. d susceptibility established by the European Union at time of manufacture FCC Warning Changes or modifications to the MiniCycler not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment A 2 Appendix MI Appendix B Warranties U S Domestic Warranty Standard RESEARCH Inc warrants NEW MJ RESEARCH BRAND THERMAL CYCLERS MODELS PTC 100 PTC 150 PTC 200 amp PTC 225 against defects in material and workmanship for a period of two years from the date of purchase If a defect is discovered MJ RESEARCH Inc will at its option repair replace or refund the purchase price of the THERMAL CYCLER at no charge to the cus tomer provided the product is returned to MJ RESEARCH Inc within the warranty period In no event will MJ REsEARCH Inc be responsible for damage resulting from accident abuse misuses or inadequate packaging of returned goods ANY implied warranties including implied warranties of the merchantability and fitness for a particular purpose are limited in duration to TWO years from the date of original retail purchase of this product The warranty and remedies set forth above are exclusive and in lieu of all others oral or written expressed or implied No MJ RESEARCH dealer agent or employee is authorized to make any modi fication addition or extension to this warranty except in the form of the extended warranty out lined below
28. dapter plate 4 7 loading into block 4 8 sealing with Hot Bonnet and caps film 4 7 with oil or wax 4 6 selecting 4 4 selection chart table 4 14 thin vs thick walled 4 5 Sealing sealing system selection chart table 4 4 Self test 4 2 Specifications 2 6 T Temperature control methods block control 6 3 probe control 6 3 Terminology iv Troubleshooting error messages 9 2 problems with machine performance 9 5 problems with protocols 9 3 Tubes See Sample vessels
29. displayed In sample Probe Control NO Yes Select Yes and press Proceed The protocol will begin running under probe control If probe control is not desired select No and press Proceed The proto col will begin running under block control Reading the Runtime Screen During a protocol run a runtime screen will be displayed QUIKSTEP 1 65 4 1 This screen lists the program name Quikstep in the example above the protocol step that is running 1 the block temperature 65 4 C and the cycle number 1 If the probe is installed the temperature of the repre sentative sample inside the probe is displayed 62 7 C CUSTOM 1 65 4 1 62 7 5 3 Bl MiniCycler Operations Manual The runtime screen will show each step of the protocol as it executes When the target temperature for a given step is reached a timer in the middle of the second line will begin running QUIKSTEP 2 92 0 1 00 10 The timer shows the length of time the samples have been held at the displayed temperature When another step begins the timer disappears until the new step s target temperature is reached At this point the timer begins running for the new step Reading the Protocol Completion Screen When the protocol ends a message is displayed QUIKSTEP 5 75 0 Complete The number of the last step in the protocol 5 in the example above and the temperature of the block at the completion of the program 75 are displayed in the up
30. e the case to open wider than this to avoid dam aging the case s hinge 4 To close the case push it down to cover the machine Replace the screw holding the case to the machine base Figure 10 1 Opening the MiniCycler case 10 2 Changing the Sample Block Mi Changing the Sample Block Follow this procedure to change the sample block 1 Unplug the power cord from the machine and open the case as de scribed above Locate the block and identify the two white ribbon cables exiting from it fig 10 2 Grasp the ribbon cables just above their board connections and gen tly pull upward to disconnect them Locate the metal latch holding the block onto the circuit board fig 10 3 Press down on the latch then pull the block toward the latch The block will slide out over the latch fig 10 4 Tip Ifyou have trouble sliding the block out insert the tip of a flat head screwdriver between the latch and the bottom of the instrument Use the screwdriver to push the latch down while sliding out the block Slide the new block into place firm pressure may be required Reconnect the ribbon cables to their pins on the board Make sure to align the pins with to the connectors correctly Close the case as described above Figure 10 2 Changing the sample block EN 10 3 MiniCycler Operations Manual Figure 10 3 R
31. ed 0 2 mL tube use the ADR 0153 adapter plate provided If the unit is programmed and plugged in correctly carefully try to deter mine if the heated lid is getting hot do not touch the inside of the lid because if it is working it will be extremely hot If the lid is cool it may be in need of repair Please call customer service at 888 MJCYCLE Selecting the Correct Sample Vessel 4 4 RESEARCH offers a full range of tubes and microplates manufactured to the specifications of each type of block to ensure a precise fit See chap ter appendix 4 A for a complete list Keep in mind that differences in tube and plate composition and wall thickness among the many brands available can affect reaction results Protocols may require some adjust ment to ensure optimum results when using a new vessel type Note certain tube and sealing options require the use of the Microseal Adapter see page 4 7 0 5mL tubes The 16 well block accepts either thick or thin walled tubes Make sure thick walled 0 5mL tubes fit the wells snugly Since these tubes were origi nally designed for centrifuges some brands may not fit tightly in ther mal cycler wells Thin walled 0 5mL tubes were specifically designed for Figure 4 2 Operation thermal cycling and the higher quality brands provide a good and con sistent fit MJ RESEARCH provides thin and thick walled 0 5mL tubes de signed for precise block fit 0 2mL tubes All types of
32. ed to change anything Minimize primer dimer production by designing primers with no 3 self complementarity Reoptimize magnesium concen tration and annealing temper ature to maximize desired pro duct and minimize primer dimers Program a hot start into the protocol Check concentrations of compo nents May need to reoptimize magnesium concentration Reoptimize annealing temperature It should be within a few degrees of the temperature for the primers using the nearest neighbor method Check extraction and purifi cation protocols Add additional purification steps if necessary Check nucleic acid preparations by gel electrophoresis 9 3 Bl MiniCycler Operations Manual Problem Cause Action No reaction products Protocol contains a wrong value Use List to check protocol s obtained temperature control method temperatures and times Reaction component omitted from Check reaction assembly proto mixture col ensuring that mixture con tains appropriate components in correct concentrations Denaturation temperature too Use 92 C for denaturation low Annealing temperature too high Check for appropriate anneal for primers ing temperatures of primers using available computer pro grams or empirical testing Probe failed causing machine to Check screen for probe failure run protocol under block control error message Probe may need servicing or replacing Call MJ RESEARCH or your local di
33. egrees Celsius to be entered for the step Type any number between 9 0 and 105 0 as the target temperature 92 5 is used in the example below Step 1 Temperature 92 5 6 5 Bl MiniCycler Operations Manual 6 6 Press Proceed The temperature will be accepted and a screen allow ing entry of an incubation time will be displayed Step 1 Hrs _ Min Sec A value must be entered for Hrs hours Min minutes and Sec sec onds Use the Select keys to move the cursor to each option and do one of the following type a number or press Proceed to automatically enter a value of 0 For example to enter an incubation time of 30 seconds press Proceed twice The value of 0 will automatically be entered for Hrs and Min and the cursor will be positioned at Sec Use the keyboard to type 30 The screen will look like this Step 1 Hrs 0 Min Sec 30 Press Proceed The times will be accepted and the Enter Menu will be displayed again Use the Enter Menu to add another step to the program Entering a GoTo step To enter a GoTo step select GoTo from the Enter Menu and press Pro ceed The first GoTo screen will be displayed Step 2 Go to step The first line of this screen shows the number of the step being pro grammed 2 is used in the example above The second line of the screen allows entry of the number of the step the program should cycle back to Type the number of the step the program should
34. eleasing the sample block latch SSS yE AL PRS ANS KT Figure 10 4 Sliding the sample block out Appendix MI Appendix Safety Warnings Warning Operating the MiniCycler before reading this manual can constitute a personal injury hazard Only qualified laboratory personnel trained in the safe use of elec trical equipment should operate this machine 4 Warning Always disconnect the power cord before opening the base of the PTC 150 Mini Cycler to avoid receiving an electrical shock 4 Warning The MiniCycler incorporates neutral fusing which means that live power may still be available inside the unit even if a fuse has blown or been removed Although the machine will not be operational you could still receive a shock if you fail to dis connect the power cord from the machine before opening the base after a fuse has blown Never open the MiniCycler s base with the power cord connected and never connect the power cord to the machine when the base is open even if a fuse has blown Warning The MiniCycler Hot Bonnet inner lid can become hot enough to inflict serious burns during the course of normal operation Use extreme caution if the Hot Bon net is removed while still hot Do not touch the inner lid when it is hot Do not place a hot MiniCycler Hot Bonnet on a flammable surface to avoid starting a fire Warning The MiniCycler s
35. enter a value of 00 for minutes or seconds sim ply press Proceed Both zeros will be entered at once For example to change the time value for the example above from 00 00 10 to 00 00 30 press the right arrow key to move the cursor to the first digit of the time value CUSTOM1 1 94 0 00 00 30 Typea zero The zero will become the first digit of the hours element and the values for minutes and seconds will disappear from the screen CUSTOM1 1 94 0 0 Press Proceed A second zero will automatically be entered for the hours element and the cursor will move to the minutes element CUSTOM1 1 94 0 00 Press Proceed Two zeros will automatically be entered for the min utes element and the cursor will move to the seconds element CUSTOM1 1 94 0 Q0 00 _ Type the number 30 then press Proceed The number will be entered 7 3 Bl MiniCycler Operations Manual 7 4 for the seconds element and the cursor will move back to the first digit for the hours CUSTOM1 1 94 0 99 00 30 Editing slope step To edit a slope step you must retype both a sign plus or minus and a number Press the period key to enter a plus sign and the hy phen to enter a negative sign Press Proceed after entering the new slope step value 8 Maintenance Cleaning the Chassis and Block 8 2 Cleaning the Air Vents 2 8 2 Cleaning Radioactive or Biohazardous Mate
36. er must be en tered for Hrs Min and Sec Press Proceed to move the cursor to the next time element When the denominator has been typed press Proceed The numbers will be accepted into the program and the Enter Menu will be displayed again Use the Enter Menu to add another step to the program Entering the End Step To enter the End step select End from the Enter Menu then press Pro ceed A confirmation screen will be displayed Step 6 End Press Proceed The End step will be entered into the program The program will be stored and the Run Enter Menu will be displayed 6 9 Bl MiniCycler Operations Manual Programming an Indefinite Hold Programs can be designed to end in an indefinite hold at a specified tem perature This allows completed reaction products to be held at a stable refrigeration temperature at the end of a run until they can be conve niently removed To program an indefinite hold program a temperature step immediately before the protocol s End step Type the desired temperature for the in definite hold then give the step an incubation time of 0 hours 0 minutes and 0 seconds When this temperature step is reached the block will heat or cool the samples to the desired temperature and hold them there until you press Proceed or manually end the program by pressing Can cel or Stop Creating a Program That Requires Manual Stepping To create a program that must be manual
37. eturn the MiniCycler for service See appendix D for shipping instructions Setting Up the MiniCycler Insert the power cord plug into its jack see fig 2 1 then plug the cord into an electrical outlet see Power Supply Requirements below Situ ate the machine according to the instructions below Environmental Requirements 32 Ensure that the area where the MiniCycler is installed meets the follow ing conditions for reasons of safety and performance Indoor nonexplosive environment Ambient temperature 4 32 C Relative humidity between 10 and 90 Protection from excessive heat e g radiators and accidental spills NT Note operation at altitudes significantly above sea level may affect operation particularly heat dissipation Installation Power Supply Requirements The MiniCycler requires 100 240 50 60Hz and a grounded outlet The machine can use current in the specified range without adjustment so there is no voltage setting switch Air Supply Requirements Air is taken in from a vent on the right side of the machine and exhausted from a vent on the left side of the machine see figs 2 1 and 2 2 If the ma chine does not get enough air or gets air that is too warm it can overheat Overheating can cause the machine to display a warning message Hleat S ink Overheating Check Air Flow or even to shut itself off To prevent overheating position the machine at least 10cm from ot
38. her thermal cyclers or walls This is particularly important if two or more MiniCyclers are used in one installation one machine can blow all of its exhaust air directly into the intake vent of its neighbor if they are placed closely side by side If the ambient air temperature exceeds 32 C use fans or adjust the air conditioning to cool the air that the machine re ceives Keep the air intake vent clean and free of obstructions e g loose sheets of paper 3 3 4 Operation Turning the MiniCycler On 4 2 Using the Control Panel 4 2 Operation keys essere 4 2 Opening and Closing the Lid 4 3 Mounting the Hot 4 3 Selecting the Correct Sample Vessel 4 4 0 5ml Tubes x Eee rette 4 4 02ml a 4 4 Microplate 4 4 Thin vs thick walled tubes 002 422 222 4 4 5 Positive displacement pipette tips and glass capillary tubes 4 6 Sealing Sample Vessels 4 6 Sealing with oil or 4 6 Sealing with the Hot Bonnet and caps or film sheets sss 4 7 The Microseal Adapter 4 7 Sealing glass capillary tubes 4 7 Sealing positive displacement pipette tips
39. hutdowns see Air Supply Requirements chapter 3 Cleaning Radioactive or Biohazardous Materials Out of the Block 8 2 When cleaning machines that have been running radioactive or biohazardous reactions consult your institution s radiation safety officer or biosafety officer regarding methods monitoring and disposing of con taminated materials If mild cleaning agents do not remove radioactivity harsher chemicals may be used Users have suggested the detergent PCC 54 Pierce Chemi cal Co Rockford Illinois Pierce Eurochemie B V Holland Micro Clean ing Solution Cole Parmer Niles Illinois and Dow Bathroom Cleaner available in supermarkets Maintenance Caution Harsh cleaning agents are corrosive to aluminum and must never be used on bare aluminum blocks MJ REASEARCH blocks are anodized so they have a protective coating of aluminum oxide Still harsh agents such as those above must be thoroughly rinsed away within a few minutes of application or the anodization will degrade Changing the Fuses The circuits in the MiniCycler are protected by two fuses 2 5TA 250V Slo blo When a fuse blows the MiniCycler immediately shuts down and cannot be turned back on The machine records the event as a power loss so if a protocol is running when a fuse blows the machine will re sume the protocol run when the fuse is replaced and power restored see Resuming a Protocol after a Power Outage chapter 5
40. icroseal A The adapter is essentially a heat conducting metal spacer it raises the clearance lower profile sample tubes that would other wise not engage the MiniCycler lid s safety interlock and it provides the lid pressure the Microseal film requires for proper sealing Loading Sample Vessels Loading tubes and plates If you will be using the Hot Bonnet with a small number of tubes load at least one empty tube in each corner of the block see Mounting the Hot Bonnet p 4 3 To ensure uniform heating and cooling of samples tubes and plates must make good thermal contact with the block Adequate contact is ensured 4 7 Bl MiniCycler Operations Manual by always following these recommendations e Check that the block is clean before loading samples see chapter 8 for instructions on cleaning block wells e Firmly press the tubes or microplate into the wells and use the heated lid or the sponge rubber pad to ensure that tubes are firmly pressed into the block Do not use the sponge rubber pad with heated block Using the sponge rubber pad to improve tube contact with the block When using the MiniCycler without the Hot Bonnet a magnetic sponge rubber pad may be attached to the lid to help press loaded tubes into the block wells Two sizes of pad are available e A 1cm pad for use with 0 5mL tubes in the 16 well block e A2cm pad for use with 0 2mL tubes or 25 well microplates Attach the magnetic backing of
41. ime required for heat to transfer to the sensor within the probe tube Therefore probe control protocols typically require incu bations that are 15 20 seconds shorter than incubations for block control protocols For example a denaturation step in a block control protocol calling for 92 for 30 sec would be reduced to 92 for 10 sec in a probe control protocol 6 3 Bl MiniCycler Operations Manual Entering a New Program Programming the MiniCycler occurs in three stages 1 Initiating the program 2 Naming the program 3 Entering the program s steps including the End step Each stage involves typing values from the keyboard or making selec tions from a menu Programs may be edited as they are being entered or after they have been stored Programs are automatically saved when the End step is entered Initiating the program To initiate anew program select Enter Program from the Run Enter Menu then press Proceed The Main Menu will be displayed _NEW List Edit Delete Select New then press Proceed The naming screen will be displayed Name A Naming the program Program names may be up to eight characters long and may consist of any combination of letters Roman and Greek numbers punctuation marks or Japanese Katakana Press the right Select key to scroll forward and the left Select key to scroll backward through the alphabets and characters avail able which are presented in this order Roman
42. ing the Oil 3 Gently place the thermistor into the new tube and snap the lid closed Make sure that the lid from the original probe tube attached to the thermistor fits on the new tube and that the tube is long enough to accommodate the probe wire The sensor should rest on the tube s bottom Caution The thermistor is extremely fragile Handle it with great care Adding the oil Viscous oils not water are the best choice for the probe tube s represen tative sample They closely mimic the thermal characteristics of buffer solution which changes temperature sluggishly due to the high specific heat of water Light and heavy mineral oil and silicone oil may be used table 4 1 MJ RESEARCH recommends using heavy mineral oil because the formula for determining the correct volume of oil to use is easy to remember and it is widely available and inexpensive But whichever type of oil you use be careful to add the correct amount of oil Loading and connecting the probe Seat the probe tube in the center of the block Plug the probe into its jack see fig 4 3 One of the pin holes in the probe jack has been blocked off so that the probe cannot be plugged in incorrectly Running a protocol under probe control To run a protocol under probe control load and connect the probe as described above Select a protocol that has been designed for probe con trol and press Proceed The following screen will be displayed In s
43. ive information on where your application fits please contact Roche ar types of enzyme or thermal cycler may require separate Addresses for Contacting Hoffmann LaRoche n the United States Kathe Kiehn licensing Manager Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda CA 94501 USA 510 814 2970 e Fax 510 814 2977 n other nations Dr Agnieszka Junosza Jankowski PCR Licensing Manager F Hoffmann La Roche ltd Building 222 350 CH 4002 Basel Switzerland 41616873031 Fax 41616872113 Note Please do not construe the information on this page as legal advice for more information please contact your institution s legal department D 1 Appendix E CE Conformity DECLARATION OF CONFORMITY MJ RESEARCH INC hereby declares that the MJ RESEARCH PTC 150 MiniCycler thermal cycler conforms to the following directives and standards APPLICATION OF COUNCIL DirEcTIVES 89 336 EEC amp 73 23 EEC STANDARDS TO WHICH CONFORMITY IS DECLARED EN55011 EN50082 1 Canadian Standards Associa tion CSA Std 1010 EMC for industrial scientific medical equipment EMC for generic immunity compliance to low voltage directive respectively TYPE OF EQUIPMENT Scientific Equipment for Laboratories NuMBER 150 YEARS OF MANUFACTURE 1997 and onward RESEARCH INC as manufacturer of the equipment described above certifies that this model has been tested and
44. kaging materials You can also request a loaner machine which will be provided if available a rental fee may apply You can use the loaner s packag ing to return the machine needing repair 4 Write the RMA number on the outside of the box 5 Ship the machine freight prepaid to the following address We recommend you purchase insurance from your shipper Ship to Repair Department MJ Research Inc 590 Lincoln St Waltham MA 02451 C1 Appendix D Appendix D PCR Licensing table PCR LICENSING INFORMATION Background The PCR process is covered by patents owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd collectively called Roche Roche has granted exclusive and non exclusive licenses for various applications of PCR and Roche and its licensees provide end user licenses within their designated fields These licenses have different terms depending on the field and different rules may apply in different countries In the U S and most other nations the user should always have proper license to perform PCR The following table reflects MJ Research s understanding of the requirements set by Roche and its licensees for particular classes of users to purchase PCR licenses in the United States This table is not endorsed by Roche and all thermal cycler users are encouraged to contact Roche for further information Authorization of thermal cyclers The Perkin Elmer Corporation holds the exclusive license fro
45. ly progressed to each step pro gram an indefinite hold for each temperature step in the protocol see Programming an Indefinite Hold above When the protocol runs you may then advance it through its steps at the time desired by pressing Proceed Editing While Entering a Program 6 10 Changing the last value entered or last menu option chosen To change the last value entered or last menu option chosen press Can cel The choice just made will be cancelled and another value may be entered or another menu option chosen Press Proceed after changing a value so that the program will accept it Changing all the values in the step being entered To change all the values in the step currently being entered repeatedly press Cancel Each time you press the key the cursor will move back ward through the values for the step deleting them as it moves When all values for the step have been deleted enter new values or press Can cel one more time to display the Enter Menu for the step At this point you can re enter the step Programming Changing values in earlier steps of a program being entered To change one or more values in earlier steps of the program you are entering repeatedly press Cancel until the Enter Menu for the step you are working on is displayed Press Cancel one more time to display the last entered step The step will be displayed in a special format CUSTOM1 3 94 0 00 00
46. m Roche to grant sublicenses to perform PCR in specific fields such as research and forensics Since 1992 Perkin Elmer has generally issued licenses in the form of a running royalty with sale of specific licensed enzymes which carry a label stating that reactions must be run in an authorized thermal cycler Research does not sell authorized thermal cyclers and believes that PE s right to charge a thermal cycler authorization fee is probably legally unenforceable in the U S this matter is under review by the U S Federal District Court However Perkin Elmer will sell such an authorization for any individual thermal cycler It may be prudent in some circumstances to authorize MJ cycler after purchase for Human amp Animal Diagnostics WITH LICENSED Test Kirst WirTH DIAGNOSTIC SERVICE LICENSES or Use Usually None see THERMAL Cycier Usually None see LICENSE actual terms of license actual terms of RESEARCH FORENSICS ETC Thermal Cycler authorized for PCR Non PCRt CYCLE SEQUENCING PRINS ETC Usually None see Usually None see ENZYME LICENSE actual terms of actual terms of N B For each column both the indicated thermal cycler license and the indicated enzyme license are This table refers to PCR licenses only other processes and particu license Enzyme with PCR label license T For definit
47. meric key pad for entering values into programs Operation keys Select keys left and right arrows Move the cursor one space or op tion to the left or right in the LCD window Proceed Accepts a selected menu or screen option Cancel Terminates a running protocol during programming or edit ing cancels the last entry Stop Terminates a running protocol Pause Pauses a running protocol Instant Programs an Instant Incubation see 5 6 Operation Opening and Closing the Lid To open the MiniCycler gently push the lid latch see fig 2 2 forward until it disengages then push the lid up To close the lid push the lid down until the latch engages with a distinct click Mounting the Hot Bonnet The Hot Bonnet mounts on two metal pins inside the retainer When the lid s lever is lowered the lid locks into place on the pins and the inner lid is lowered onto the tops of loaded sample vessels fig 4B Certain models of the MiniCycler lack these pins A retrofit to install them is avail able Contact your distributor or MJ Research for further information Follow this procedure to mount the Hot Bonnet 1 Make sure the Hot Bonnet is the correct size for the block The sticker on the side of the unit identifies it by catalogue number HBA 1151 is for 16 well blocks HBA 1152 is for 25 well blocks 2 If only a few tubes are being run make sure one tube is loaded into each corner of the block
48. metal adapter Contact RESEARCH or your distributor Make sure power cord is plugged into machine correctly Make sure electrical outlet is operational Check fuses See po Reset the microprocessor by turning machine off then back on while pressing the Cancel key at the same time If problem persists contact MJ RESEARCH or your distributor 9 5 10 Changing the Sample Block Opening and Closing the MiniCycler s 10 2 Changing the Sample Block 10 2 10 1 Bl MiniCycler Operations Manual Caution Avoid changing the sample block frequently The cables and connectors are fragile and can be damaged in as few as 50 100 uses Opening and Closing the MiniCycler s Case Follow this procedure to open and close the MiniCycler s case 1 Unplug the power cord from the machine Put the power switch in the 0 position 2 Using a Philips head screwdriver remove the large black screw on the left side of the machine fig 10 1A 3 Place the tip of a finger under the edge of the case just beneath the screw hole and pull outward and upward on the case The case latch will release and the case can then be lifted up and over to reveal the interior of the machine 4 Warning Do not plug the power cord into the machine while the case is open You could receive a serious shock Caution case is designed to open to 90 angle Do not forc
49. n bare aluminum blocks MJ RESEARCH blocks are anodized so they have a protective coating of aluminum oxide Still harsh agents such as those above must be thoroughly rinsed away within a few minutes of application or the anodization will degrade 4 13 Running a 1 5 2 Running a protocol with the Hot Bonnet installed 5 2 Running a protocol under probe control 5 3 Reading the Runtime Screen 5 3 Reading the Protocol Completion Screen 5 4 Manually Stepping Through a Protocol 5 4 Pausing a Running Protocol 5 5 Stopping a Running Protocol 5 5 Resuming a Protocol after a Power Outage 5 5 Running an Instant Incubation 5 6 Running Protocols 5 1 Bl MiniCycler Operations Manual Running a Protocol 5 2 Either a custom designed protocol or one of the factory installed proto cols may be run See appendix F for descriptions of the factory installed protocols which may be edited to fit your needs To run a protocol select Run Program from the Run Enter Menu then press Proceed If protocols have been stored in the machine they will be displayed Press one of the Select keys until the name of the protocol you wish to run is displayed Press Proceed again The protocol will begin r
50. ocol will complete the step that was paused and continue on to the next step Stopping a Running Protocol Press Stop or Cancel to stop a running protocol The program will stop running and the Run Enter Menu will be displayed again Note Turning off the machine will not stop a running protocol Instead the MiniCycler will assume that a power outage has occurred and will resume running the protocol when the machine is turned on again see below 5 5 Bl MiniCycler Operations Manual Resuming a Protocol after a Power Outage If a power failure occurs when a protocol is running the MiniCycler will hold the protocol in memory for at least 24 hours and sometimes up to 10 days The exact duration depends on environmental conditions When power is restored the protocol will begin running again at the point at which it was stopped When the protocol ends the protocol completion screen will be displayed with a special notice on its last line QUIKSTEPCOMPLETE Press Proceed Press Proceed as instructed A screen about the power outage will be displayed The screen will identify the step and the cycle that were run ning when the power failure occurred AC POWER FAILED Cyc 3 Step 2 Press Proceed to remove this screen The Run Enter Menu will be dis played again Running an Instant Incubation 5 6 The MiniCycler may be used as a constant temperature incubator by pressing Instant the zero key while the
51. oplate 100 240VAC rms no adjustment needed among voltages within these ranges 50 60Hz single phase 250W maximum Two T2 5A 250V One 2 x 16 LCD alphanumeric display One 25 pin 8 bit parallel interface printer port optional 80 typical programs in nonvolatile memory each program may have up to 100 steps maximum of 400 steps among all programs 3 0kg 22 x 28 x 11cm high Installation Packing 54 5 3 2 Setting Up the MiniCycler 3 2 Environmental Requirements 3 2 Power Supply Requirements 3 3 Air Supply 3 3 3 1 Bl MiniCycler Operations Manual Packing List After unpacking the MiniCycler check to see that you have received the following One MiniCycler with the sample block of your choice One power cord Two fuses A 1cm thick magnetic sponge rubber pad 16 well block or a 2cm thick pad 25 well block One red metal adapter plate 25 well block with heated lid only The PTC 150 MiniCycler Operations Manual this document Warranty registration card and extended warranty application US customers only If any of these components are missing or damaged contact MJ REsEARCH or the authorized distributor from whom you purchased the MiniCycler to obtain a replacement Please save the original packing materials in case you need to r
52. per right hand corner of the screen The temperature reading will change as the block cools to ambient temperature Press Proceed to remove this screen The Run Enter Menu will be dis played and another protocol may be run Manually Stepping Through a Protocol 5 4 A running protocol can be manually advanced through its steps As soon as a step has reached its target temperature i e when the timer begins running for the step press Proceed to progress the protocol to its next programmed step The next step will immediately be displayed Running Protocols Note Pressing Proceed while a step is ramping will have no effect A step must have reached its target temperature before the protocol can be manually advanced to the next step Protocols can be programmed to require manual stepping see p 6 10 Pausing a Running Protocol Press Pause to temporarily stop a running protocol If Pause is pressed during temperature ramping the protocol will pause as soon as the target temperature is reached and samples will be held at the dis played temperature If Pause is pressed after a step has reached its target temperature the protocol will immediately begin holding samples at the current incubation temperature The timer will stop running and the word Pause will be displayed QUIKSTEP 2 92 0 9 PAUSE To resume the protocol press Pause again The timer will begin run ning again and the prot
53. r e Chapter 8 explains the proper maintenance of the MiniCycler e Chapter 9 offers troubleshooting information for the MiniCycler e Chapter 10 describes how to change the MiniCycler s block Important Safety Information Safe operation of the MiniCycler begins with a complete understanding of how the machine works Please review this entire manual before at tempting to operate the MiniCycler Do not allow anyone who has not reviewed this manual to operate the machine The MiniCycler can generate enough heat to inflict serious burns and can deliver strong electrical shocks if not used according to the instruc tions in this manual Please read the safety warnings and guidelines in appendix B and exercise all precautions outlined in them Front View Fig 2 1 2 2 Back View Fig 2 2 2 2 Control Panel Fig 2 3 00 0 0 2 3 Blocks 2 4 Accessories eee eee eeeeesesss s 2 4 Hot Bonnet Fig 2 4 esee 2 4 Temperature Probe Fig 2 5 2 5 Sponge Rubber Pads Fig 2 6 2 5 Specifications doeet ERE ere 2 6 Layout and Specifications 2 1 Bl MiniCycler Operations Manual Front View Fig 2 1 Air intake vent Power cord jack Power switch Lid latch Air exhau
54. r 30 sec 1 92 for 30 sec 2 60 for min 2 60 for3 min 3 92 for 30 sec 3 GoTo step 1 29 times i e cycle 4 60 for 3 min back to step 1 and repeat steps 5 92 for 30 sec 1 and 2 29 more times 6 60 for 3 min 4 End 7 92 for 30 sec continues for total of 60 lines Choosing a temperature control method The MiniCycler can control block temperature in two ways each of which has different implications for the speed and accuracy of sample heating Block control The MiniCycler adjusts the block s temperature to maintain the block at programmed temperatures independent of sample temperature Probe control The MiniCycler adjusts the block s temperature to maintain an in sample probe at programmed temperatures Block control Under block control the temperature of samples always lags behind the temperature of the block The duration of this time lag depends on type of sample vessel and sample volume but typically is between 10 and 30 seconds Probe control Probe control is available for MiniCyclers equipped with the 16 or 25 well block Special care must be taken to fill the probe with the correct amount of oil and to seat the probe and the samples correctly Otherwise actual sample temperatures can vary widely from the probe s tempera ture Probe control cannot be used with microplates or slides Under probe control the machine will slightly overshoot its heat targets to account for the t
55. rams and press Proceed Each program stored in the machine will be printed The MiniCycler parallel printer port is compatible with Epson LX 810 dot matrix printers and many other IBM compatibles Only printers that support the Epson extended graphics set will be able to print the de gree character Other printers will print an x instead of the degree character 6 13 Initiating Editing ccccccccccsccscccccccsccsscees 7 2 Editing the Program 7 2 Editing temperatures and cycling values 7 2 Editing a time value 7 3 Editing slope step 2 2 1 7 4 Editing Programs 7 1 Bl MiniCycler Operations Manual Initiating Editing To edit an existing program the machine must be in Edit mode To enter Edit mode select Enter Program from the Run Enter Menu and press ceed gt The Main Menu will be displayed Select Edit from the Main Menu and press Proceed A screen similar to the following will be displayed Edit CUSTOM1 The machine is now in Edit mode Use the Select keys to select the program you wish to edit When the name of the desired program is dis played press Proceed The first step in the program will be displayed CUSTOM1 1 94 0 00 00 20 programmed temperature for this step is 94 0 C The incubation time is displayed in the form of 00 00 00 The first two digits represent hour
56. rials Out of the Block 8 2 Changing the FUSES 8 3 Bl MiniCycler Operations Manual Cleaning the Chassis and Block Clean the outside of the MiniCycler with a damp soft cloth whenever something has been spilled on it or the chassis is dusty A mild soap solu tion may be used if needed Clean the block s wells whenever anything is spilled into them or when the oil in them is discolored or contains particulate matter It is particu larly important to prevent the buildup of old dirty oil which will inter fere with vessel seating and diminish thermal coupling of sample vessels to the block Clean the wells with a swab moistened with water 95 ethanol or if a thorough cleaning is needed a 1 100 dilution in water of 5 25 0 7M so dium hypochlorite household bleach If using sodium hypochlorite swab the wells with water afterward to remove all traces of it Do not clean the block with caustic or strongly alkaline solutions e g strong soaps ammo nia sodium hypochlorite at a higher concentration than specified above These can damage the block s protective anodized coating 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 If these vents become clogged with dust and debris airflow to the MiniCycler s heat sink is hampered eventually causing overheating and s
57. s the second two digits represent minutes and the last two digits represent sec onds The step in the example is programmed for a 20 second incubation Editing the Program 7 2 Press the left Select key to scroll backward through the program steps and the right Select key to scroll forward As the left or right Select key is pressed the cursor will progressively move backward or forward through each value in a step and then back to the preceding or forward to the following step Editing temperatures and cycling values To change a temperature or a cycling value position the cursor on it and type the new value Press Proceed The new value will be accepted and the cursor will move forward to the next value in the step To cancel a change press Cancel The just typed value will disappear and a blank line will be displayed in its place Type a new value and press Proceed Editing Programs Note Becareful when editing program values Once a value is changed or deleted you cannot make it reappear Editing a time value To edit any part of a time value you must retype the number for each element in the time value hours minutes and seconds Press Proceed after typing each time element The number just typed will be accepted and the cursor will move to the next time element To enter a value of 00 for hours type a zero then press Proceed The second zero will auto matically be entered To
58. st vent Control Panel Fig 2 3 Step Cycle Time Sample MJ RESEARCH Layout and Specifications 2 3 Bl MiniCycler Operations Manual Blocks Available 16 well block holds 16 x 0 5mL tubes 25 well block holds 25 x 0 2mL tubes or one 25 well V bottom microplate Accessories Hot Bonnet Fig 2 4 Available for 16 and 25 well blocks Locking lever ___ Pin slot 2 4 Layout and Specifications Temperature probe Fig 2 5 Allows machine to control reactions according to a representative sample s tem perature Available for 16 and 25 well blocks Magnetic sponge rubber pads Fig 2 6 Used to help press tubes or a plate into the block Available for use with 16 and 25 well blocks Thin 1 pad Thick 2cm pad for 16 well block for 25 well block 2 5 Bl MiniCycler Operations Manual Specifications 2 6 Thermal range Accuracy Thermal homogeneity Sample capacity Line voltage Frequency Power Fuses Displays Ports Memory Weight Size 9 to 105 C no more than 35 C below ambient temperature 0 3 C of programmed target at 90 C NIST traceable 0 4 C well to well within 12 seconds of arrival at 90 C 0 3 C well to well within 40 seconds of arrival at 90 C 16 well block 16 x 0 5mL tubes 25 well block 25 x 0 2mL tubes or one 25 well micr
59. stri butor Probe not filled with correct Fill probe tube with correct amount of oil amount of oil see p 4 11 Reaction mix contains an inhibitor Spike a complete reaction mix e g heme from blood with a control template and primer set Reaction vessels not making good Use only high quality thermal contact with sample tubes plates that fit block block snugly Ensure that wells are free of foreign materials that would interfere with tube plate seating 9 4 Troubleshooting Problems Related to Machine Performance Problem Beeper sounds continu ously MiniCyclers with Hot Bonnet only Block does not heat or cool Machine will not power up Machine starts in middle of a protocol when turned on Strange characters appear in LCD window or text in window freezes or scrolls repe titively Cause Hot Bonnet is not plugged into the machine Tubes are not in all 4 corners of the block so lid is tilted A microplate or flat capped tubes are being used Heat pump or fans are malfunctioning Power cable is not plugged into machine electrical outlet is dead or fuse has blown Machine was turned off before protocol run had finished LCD window or machine microprocessor is malfunc tioning Action Cancel protocol Plug Hot Bon net cable into its port at back of machine and restart protocol Put tubes empty if necessary in all 4 corners of the block Need to use red
60. tand 5Specificallons eisa a 2 1 3 Installato RN 3 1 A VO perattOn sere nt haem ner mU BA 4 1 5 Running Protocols AR a aah uci Reena etait 5 1 o Creating Pro oats 6 1 74 Editing RO PATS aseo e epp beide prelati Gs qa titia 7 1 deett than l ERES LR ex SEE 8 1 9 a a RE ARTE 9 1 10 Troubleshooting 10 1 Appendix Safety Warnings and Guidelines A 1 Appendix B Warranties sssi eadi cre uet teg ef gu o ido bu d Pe Cose 1 Appendix C Shipping Instructions for US Residents C 1 Appendix Licensing Table odii EU D 1 Appendix E Declaration of CE Conformity ala petii e E 1 Appendix F Factory installed Protocols cette tt Pea va eps Reve saran F 1 DIET Su Ea In 1 Documentation Conventions Typographic Conventions The names of keyboard keys are in sans serif type and placed within double angle brackets Example Proceed Items in programming menus are italicized Example Select Edit from the Main Menu Graphic Conventions Terminology The programming screens displayed in the LCD window are represented by a
61. thin walled 0 2mL tubes may be used in the 25 well block MJ RESEARCH sells high quality 0 2mL tubes in a number of styles includ ing individual tubes and strips Microplates V bottom polycarbonate or polypropylene microplates may be used in the 25 well block 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 10uL Polypropylene microplates and compatible Microseal A film mats or strip caps for sealing are available from RESEARCH See Sealing with the Hot Bonnet and Caps or Film Sheets p 4 7 for a description of Microseal A Ramp rate enhancement with thin walled tubes To compare ramp rates of sample tempera ture 50uL samples of water were cycled 0 5mL thin and thick walled tubes in a 200 thermal cycler with a 60 well block Thermal profile 94 C 120 sec 62 C 120 sec Data are shown for the third cycle The cycler was run under Block Control and the Hot Bonnet was heated to 105 C and tightened to a half turn past the touch point The samples in thin walled tubes reached the target temperature more quickly In this example over 30 sec onds could be saved per cycle by using thin walled tubes A comparable ramp rate en hancement can be expected when using thin walled
62. unning A 5 QUIKSTEP Running a protocol with the Hot Bonnet installed Select a protocol to run as described above and press Proceed The following screen will be displayed Use Heated Lid _YES No Select Yes and press Proceed The Hot Bonnet will begin its 99 second preheat to 108 C A screen showing a timer that counts down the seconds of this preheat period will be displayed QUIKSTEP Preheat time 89 When the lid has fully preheated the protocol will begin to run If the Hot Bonnet is removed when it is preheating if tubes are not present in all corners of the block or if the Hot Bonnet cable is disconnected from the machine the MiniCycler will begin beeping and the following mes sage will be displayed Check Bonnet Placement Running Protocols When the Hot Bonnet is replaced in the well or tubes are placed in all corners of the block or the cable is connected to the machine the Hot Bonnet will stop beeping and re start its 99 second preheat Note If the Hot Bonnet is removed when a programmed protocol is running the MiniCycler will begin beeping but no message will be dis played Running a protocol under probe control To run a protocol under probe control load and connect the probe see p 4 12 Select a protocol that has been designed for probe control Do not use probe control to run a protocol designed for block control Press Pro ceed The following screen will be
63. vessels which prevents condensation refluxing and changes in reaction concentrations The lid also exerts pressure on the tops of vessels loaded into the sample block helping to maintain a va por tight seal and to firmly seat the vessels in the block Caps film or mats must be used along with the Hot Bonnet to prevent evaporative losses Tight fitting caps are recommended for minimal va por loss and for long term storage of reaction products For optimal sealing use the Easy Cap Tool 1000 to firmly seat the caps in the tubes Microseal A film may be used when a pristine sealing surface is required for each run When a reusable sealer is appropriate the 96 well Microseal rubber sealing mats are easy to use and effective Microseal A and M sealers are sized for an array of 96 0 2mL tubes and must be cut to fit the 25 well block Follow the manufacturer s instructions when applying caps films and mats Note After a hold at 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 finished The Microseal Adapter Certain tube and sealing options require the use of the Microseal Adapter ADR 0153 This device is to be used in conjunction with flat cap tubes an array of capless 2mL tubes or a 25 well Multiplate sealed with M

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