BIO RAD Tetrad 2 Operation Manual
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BIO RAD Tetrad 2 Operation Manual

Dna engine. peltier thermal cycler
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DNA Engine Tetrad
2
®
Peltier Thermal Cycler
Operations Manual
Version 2.0
PTC-0240
DNA Engine Tetrad 2 Cycler

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Summary of Contents for BIO RAD Tetrad 2

  • Page 1 DNA Engine Tetrad ® Peltier Thermal Cycler Operations Manual Version 2.0 PTC-0240 DNA Engine Tetrad 2 Cycler...
  • Page 2 DNA Engine Tetrad ® Peltier Thermal Cycler PTC-0240 DNA Engine Tetrad 2 Cycler Operations manual Supports software version 4.0...
  • Page 3 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, 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.

  • Page 4: Table Of Contents

    Table of Contents Explanation of Symbols ..........iv Safety Warnings and Guidelines .

  • Page 5: Explanation Of Symbols

    Warning: 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.
  • Page 6 Safe Use Guidelines The DNA Engine Tetrad 2 cycler is designed to be safe to operate under the following conditions: • Indoor use • Altitude up to 2000 m • Ambient temperature 5–31°C • Relative humidity 10–90%, noncondensing • Transient overvoltage per Installation Category II, IEC 664 •...

  • Page 7: Documentation Conventions

    Documentation Conventions Before describing the various features of the DNA Engine Tetrad 2 cycler, let’s define some “common ground” conventions. • << >> will be used to indicate actual keys on the control panel, such as <<ENTER>>, <<1>> and <<LEFT>>.

  • Page 8: Chapter 1: Introduction

    Meet the DNA Engine Tetrad 2 Cycler ........
  • Page 9 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.
  • Page 10 Introduction 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.
  • Page 12 DNA Engine Tetrad 2 Cycler Control Panel ........2-2...

  • Page 13: Dna Engine Tetrad 2 Cycler Control Panel

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual DNA Engine Tetrad ® 2 Cycler Front View (Figure 2-1) Alpha unit bay ™ NEXT BACK Air intake vents Display screen Numeric keypad DNA Engine Tetrad 2 Cycler Control Panel (Figure 2-2)

  • Page 14: Dna Engine Tetrad 2 Cycler Back View

    Layout and Specifications DNA Engine Tetrad 2 Cycler Back View (Figure 2-3) RS-232 port Ethernet port Power switch Fuses Power cord jack DNA Engine Tetrad 2 Cycler Bottom View (Figure 2-4) USB A port for mouse USB B port for...

  • Page 15: Compatible Alpha Units

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual Compatible Alpha Units Single-Block Models 60 Single: Holds 60 x 0.5 ml tubes 96 Single: Holds 96 x 0.2 ml tubes or one 96-well microplate 384 Single: Holds one 384-well microplate Flat Block:...

  • Page 16: Dna Engine Tetrad 2 Specifications

    Layout and Specifications DNA Engine Tetrad 2 Specifications 0–105°C, but no more than 30°C below ambient Thermal range: temperature (10–105°C for the Slide Chambers unit) +0.3°C of programmed target at 90°C, NIST-traceable Temperature accuracy: (+0.4°C for dual Alpha units) +0.4°C well-to-well within 30 seconds of arrival at Temperature uniformity: 90°C (+0.5°C for dual Alpha units)
  • Page 18 Setting Up the DNA Engine Tetrad 2 Cycler ....... . .

  • Page 19: Unpacking And Moving The Dna Engine Tetrad 2 Cycler

    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.

  • Page 20: External Mouse Device

    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.

  • Page 21: 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.

  • Page 22: Ensuring That Air Is Cool Enough

    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.g., nearby equipment running, window...

  • Page 23: 384-Well Microplate Specifics

    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.
  • Page 24 Turning the DNA Engine Tetrad 2 Cycler On ....... . .

  • Page 25: Turning The Dna Engine Tetrad 2 Cycler On

    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.

  • Page 26: Chapter 4: Operation

    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 • The display screen is a 1/4 size VGA screen for displaying thermal cycler condi- tions and programs.

  • Page 27: Block Status Lights

    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 these ports.

  • Page 28: Removing An Alpha Unit

    3. 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.

  • Page 29: Opening An Alpha Unit

    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.

  • Page 30: 0.5 Ml Tubes

    Operation 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.

  • Page 31: Sealing Sample Vessels

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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.

  • Page 32: Adjusting The Hot Bonnet Lid's Pressure

    Operation 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.

  • Page 33: Loading Sample Vessels Into The Block

    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. Do not turn the thumbwheel more than three-quarters of a turn. This can...
  • Page 34 Operation If you use oil in the block, use only mineral oil. Never use silicone oil. Caution: It can damage the Alpha unit. One exception to this recommendation involves the use of volatile radioactive nucleotides. A small amount of oil in the block can help prevent escape of these compounds.

  • Page 35: Appendix 4-A: Tube, Microplate, And Sealing System Selection Chart

    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. 4 Reaction vessel fits block/sealing option fits reaction vessel without modification. # Reaction vessel/sealing option can be cut to fit.

  • Page 36: Appendix 4-B Safety Warning Regarding Use Of S Nucleotides

    Operation Appendix 4-B Safety Warning Regarding Use of S Nucleotides Some researchers have experienced a problem with radioactive contamination when using S in thermal cyclers. This problem has occurred with all types of reaction vessels. The Problem When S nucleotides are thermally cycled, a volatile chemical breakdown product forms, probably SO .
  • Page 37 DNA Engine Tetrad 2 Thermal Cycler Operations Manual The Solution 1. Substitute the low-energy beta emitter P 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.
  • Page 38 Creating Programs Front Panel Setup ............5-3 Display Screen .
  • Page 39 DNA Engine Tetrad 2 Thermal Cycler Operations Manual Entering Program Steps ..........5-14 The Status Window .

  • Page 40: Front Panel Setup

    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.

  • Page 41: Numeric Keypad

    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.
  • Page 42 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 ™...

  • Page 43: Types Of Programs

    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.

  • Page 44: Advanced Programs

    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.

  • Page 45: Let's Start With An Example

    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.

  • Page 46: Considerations During Program Creation

    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 samples to the desired temperature more quickly.

  • Page 47: Block Control

    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;...

  • Page 48: Choosing A Lid Control Mode

    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.

  • Page 49: Choosing A Thermal Gradient

    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.

  • Page 50: Beyond The Example Protocol: Other Considerations

    Creating Programs 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.

  • Page 51: 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.

  • Page 52: Entering A Program Using Graphical Mode

    The menu bar at the top of the Status window includes four submenus: <Programs>, <Utilities>, <Command>, and <View>. 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 <Programs>...

  • Page 53: Using The Mode Selection Window

    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 temperature and lid control modes. The current mode of temperature control is listed in the Control Mode field.

  • Page 54: Using The File Save As Window

    Creating Programs • Enter 100 from the numeric keypad. Place the cursor in the below field and select the field. We have decided to turn the lid off when the block drops below 30°C. • Enter 30 from the numeric keypad. •...
  • Page 55 DNA Engine Tetrad 2 Thermal Cycler Operations Manual • Select <New Folder>. 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.

  • Page 56: Folder Passwords

    Creating Programs Folder Passwords 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 <Edit Password> button. •...

  • Page 57: The Graphical Programming Window

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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.

  • Page 58: Deleting A Step

    Creating Programs 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 <Delete Step> 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.
  • Page 59 DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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.
  • Page 60 Creating Programs 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. Step 1 temperature step Recall that the maximum programmable temperature is 105.0°C and the minimum programmable temperature is 0.0°C in a graphical program.

  • Page 61: Entering A Gradient Step

    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 temperature step Entering a Gradient Step Step three of our program is a gradient step designed to determine the optimal annealing temperature of our oligonucleotide.

  • Page 62: Entering A Goto Step

    Creating Programs • 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.

  • Page 63: Entering A Forever Incubation

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual Entering a Forever Incubation Step 5 of our program is a forever incubation at 10.0°C to help maintain the integrity of our samples until they can be processed. Please note that the instrument can maintain samples at lower temperatures if desired (e.g., 4°C)—but, colder temperatures require...

  • Page 64: Entering A Program Using Advanced Mode

    Creating Programs Entering a Program Using Advanced Mode • Select <Programs>. Drop-down submenus appear, including <Open>, <New>, <Copy>, <Move>, <Delete>, <Delete Folder>, and <New Folder>. • Select <New>. An additional menu appears allowing you to choose <Advanced Mode> or <Basic Mode>. •...

  • Page 65: Entering A Temperature Step

    Lid Control Mode: Constant at 100°C 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.
  • Page 66 Creating Programs Gradient from 45°C to 65°C for 3 minutes Goto step 1, 29 more times An incubation at 10°C forever 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.
  • Page 67 DNA Engine Tetrad 2 Thermal Cycler Operations Manual • Similarly, enter 1 in the Time:Min field. Additionally, we want the cycler to beep after reaching the target temperature. • Select Beep on Target. At this point, in the Temperature Step window, all selections have been made according to our protocol.

  • Page 68: Entering A Gradient Step

    Creating Programs 1. Incubate at 94°C for 00:01:00 Beep on Target 2. Incubate at 92°C for 00:00:30 Beep on Target The 92°C denaturation is the first step in the cycling portion of our program. The next step is the gradient step. •...
  • Page 69 DNA Engine Tetrad 2 Thermal Cycler Operations Manual The newly created gradient can be previewed graphically. • Select <Preview>. This distribution of temperatures specified by the gradient should be reviewed and any changes to the gradient limits made before a program run. Please note that the gradient temperature differential is not linear, with a broader spread in temperature between the center columns of wells.

  • Page 70: The Extend Time Option

    Creating Programs The Extend Time Option This programming option progressively extends an incubation step with each subsequent cycle. This is typically used during an extension step, to allow for diminishing activity of an enzyme, or to allow an enzyme to do its job among an ever-increasing quantity of product.

  • Page 71: Entering A Goto Step

    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.

  • Page 72: Entering A Lid Control Step

    When using the Moto Alpha unit motorized 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 <Lid>...

  • Page 73: The Increment Temp Option

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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”...
  • Page 74 Creating Programs The final program should appear as follows: Temperature Control Mode: Calculated Lid Control Mode: Constant at 100°C 1. Incubate at 94°C for 00:01:00 Beep on Target 2. Incubate at 92°C for 00:00:30 Beep on Target 3. Gradient from 45°C to 65°C for 00:03:00 4.
  • Page 75 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.
  • Page 76 Managing and Editing Programs Opening a Program ............6-2 Opening a Program in Advanced Mode .

  • Page 77: Opening A Program

    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.

  • Page 78: Chapter 6: Managing And Editing Programs

    Managing and Editing Programs We had previously saved ADV#1 in the FOLDER2 folder. • Select the folder FOLDER2. • Select the program ADV#1. • Select <OK>. 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.

  • Page 79: Opening A Program In Basic Mode

    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-...

  • Page 80: Editing A Graphical Program

    Managing and Editing Programs 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.

  • Page 81: Editing An Advanced Program

    • Selection can also be done via the left mouse button. 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.

  • Page 82: Inserting A Step

    Managing and Editing Programs Inserting a Step Now we will insert an annealing temperature step. • Highlight step 2. Recall that new steps are inserted AFTER the highlighted step. • Select <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.

  • Page 83: File Utilities

    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.

  • Page 84: Copying A Program

    Managing and Editing Programs Copying a Program • Select <Programs> in the Status window. • Select <Copy> from the drop-down menu. 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.

  • Page 85: Deleting A Program

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual Deleting a Program • Select <Programs> in the Status window. • Select <Delete> from the drop-down menu. 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.

  • Page 86: Moving A Program

    Managing and Editing Programs Moving a Program • Select <Programs> in the Status window. • Select <Move> from the drop-down menu. Moving is the same as copying (described above), with one distinction: only one copy of the program is maintained in the To Folder. The copy in the From Folder is deleted. You will not be prompted with a verification step for this move, so exercise some caution.
  • Page 88 Running Programs Using the Instant Incubation Feature ........7-2 Running Programs .

  • Page 89: Using The Instant Incubation Feature

    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 ™...

  • Page 90: Chapter 7: Running Programs

    Running Programs • Select the Temperature field and enter the desired temperature. • 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). •...

  • Page 91: Running Programs

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual Running Programs Note: The Operations Chapter (Chapter 4) provides information on running programs with respect to the operation and use of Alpha units. For example, a program containing a gradient step will only run in 96-well Alpha units. Please review this chapter for additional technical detail, to ensure that the program you load and run is an appropriate match for your Alpha units.

  • Page 92: The Run Program Window

    <OK> 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.

  • Page 93: During The Run

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual During 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.
  • Page 94 Running Programs 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.

  • Page 95: Terminating A Run

    DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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 <Stop>. Alternatively, select the <Command> menu in the Status window, and then select <Stop> and either <All> to terminate all pro- grams running on all blocks, or select the desired block from the drop-down list.

  • Page 96: Running Multiple Programs

    Running Programs 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 run in one Alpha unit, while a typical experiment without a gradient can be run in another Alpha unit, simultaneously.
  • Page 98 Using the Utilities <About> ............. . .8-2 <Control>...

  • Page 99: About

    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 <Utilities> submenu will be discussed. The <Utilities> 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.

  • Page 100: Control

    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 COMM2 on a PC, or the serial port on a Mac. The cycler is now ready to be run in remote mode.

  • Page 101: Config

    Select <OK> to exit from this screen. <Set Date/Time> Choosing this option will allow the setting of the DNA Engine Tetrad 2 cycler’s time. Enter the appropriate information in the fields provided and select <OK>. Tech Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com...

  • Page 102: Bay Configuration

    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”...

  • Page 103: Network

    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.

  • Page 104: Gradient Calc

    “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.
  • Page 105 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.
  • Page 106 Cleaning the DNA Engine Tetrad 2 Cycler ........
  • Page 107 DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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.
  • Page 108 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.
  • Page 110 Troubleshooting Sources of Problems ...........10-2 System Problems .

  • Page 111: Sources Of Problems

    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.

  • Page 112: Error Messages

    DNA Engine Tetrad 2 cycler’s <Error Log>. 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 <Remote> section in Chapter 8 for more information on remote mode).
  • Page 113 DNA Engine Tetrad 2 Thermal Cycler Operations Manual Remote Error Code Reason Action Error Code The heat sink temperature is reg- Please observet he following istering somewhat higher than guidelines: 1) place instruments at normal. Power levels will be least 10 cm apart, 2) avoid...
  • Page 114 Troubleshooting Remote Error Code Reason Action Error Code 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.
  • Page 115 DNA Engine Tetrad 2 Thermal Cycler Operations Manual Remote Error Code Reason Action Error Code The power supply temperature was Please observet he following higher than normal. To prevent guidelines: 1) place instruments at instrument damage, the program least 10 cm apart, 2) avoid was automatically stopped.
  • Page 116 Troubleshooting Remote Error Code Reason Action Error Code A program containing a gradient If you place a 96-well Alpha unit step can only be run on a 96-well in this quadrant, you may run this None Alpha unit. gradient program. The program entered may only be No action required.

  • Page 117: Problems In Power-Up

    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.

  • Page 118: 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.

  • Page 119: Problems With An Alpha Unit

    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.
  • Page 120 Troubleshooting Problem Cause Action No reaction products Wrong protocol used. Re-run reaction using correct obtained. protocol. Protocol contains a wrong value. Use List utility to check protocol’s temperature control method, tem- peratures, and times. Reaction component omitted from Check reaction assembly protocol, mixture.
  • Page 121 DNA Engine Tetrad 2 Thermal Cycler Operations Manual Table 10-2: Protocol Difficulty Error Message Cause and Result Action Reaction is working, but “Primer-dimer” material often pro- If obtaining appropriate reaction broad low-molecular duces a broad band in the 100 bp product/s, no need to change <...

  • Page 122: Problems Due To Environmental Conditions, Setup, And Maintenance

    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.

  • Page 124: Appendix A: Warranties

    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.
  • Page 126 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.
  • Page 127 DNA Engine Tetrad 2 Thermal Cycler Operations Manual 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.

  • Page 128: Appendix B: End-User And License Agreement

    Appendix B: End-User Agreement and License (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.
  • Page 129 DNA Engine Tetrad 2 Thermal Cycler Operations Manual (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 provisions of this License Agreement.
  • Page 130 Appendix B: End-User Agreement and License 6.1 Term. The term of this AGREEMENT and the license granted hereunder shall com- mence upon first use of the CYCLER or first 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;...

  • Page 132: Appendix C: Factory-Installed Protocols

    Appendix C: Factory Installed Protocols 2-step Fast PCR protocol using Standard 2-step PCR protocol iTaq polymerase ™ STD-2 Step Temp Time iTAQ-FST 4:00 Step Temp Time GOTO 2, 29X GOTO 2, 34X Standard 3-step PCR protocol 2-step PCR protocol using iProof ™...
  • Page 133 DNA Engine Tetrad 2 Thermal Cycler Operations Manual 3-step nested primer protocol 2-step RT PCR protocol NEST PR3 RTPCR-2 Step Temp Time Step Temp Time 60:00 4:00 5:00 GOTO 3, 39X GOTO 2, 39X 7:00 7:00 3-step RT PCR protocol...

  • Page 134: Index

    Index 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...
  • Page 135 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...
  • Page 136 Index 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 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 Editing a program advanced 6-6–6-8...
  • Page 137 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...
  • Page 138 Index 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...
  • Page 139 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...
  • Page 140 Index 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 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...
  • Page 141 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.
  • Page 142 Index graph window 7-6 status window 7-7 moving 6-11 opening 6-2–6-4 advanced mode 6-2–6-3 basic mode 6-4 running 7-4 run program window 7-5 running multiple 7-9 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...
  • Page 143 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 Safety general instructions 1-3 Use of S Nucleotides 4-13 Sample vessels ensuring good thermal contact 4-10 loading into block 4-10...
  • Page 144 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...
  • Page 145 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 Uniformity. See Thermal uniformity...

  • Page 146: Declaration Of Conformity

    Declaration of Conformity Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, California, 94547, U.S.A., declares that the product PTC-0240, The DNA Engine Tetrad 2 Thermal Cycler ® to which this declaration relates, is in conformity to the following standards or normative documents. IEC61010-1 EN61326: CLASS A following the provisions of the 73/23/EEC, 89/336/EEC &...
  • Page 148 Technical Service: Call your local Bio-Rad office, or in the U.S. call 1-800-4BIORAD (1-800-424-6723). Bio-Rad Laboratories, Inc. Life Science 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 Group 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...

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