T100 Thermal Cycler Instruction Manual Rev B

Copyright ©2011 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is prohibited without written permission of Bio-Rad Laboratories, Inc.

LICENSE NOTICE TO PURCHASER This base unit, Serial No. ____________, conveys a limited non-transferable immunity from suit for the purchaser’s own internal research and development and for use in applied fields under U.S. Patent No. 5,475,610 (claims 1, 44, 158, 160-163 and 167 only), or corresponding claims in their non-U.S. counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5’ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA. This product is covered by one or more of the following U.S. patents, their foreign counterparts, or their foreign patents pending, owned by Eppendorf AG: US Patent Nos. 6,767, 512 and 7,074,367.

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T100 Thermal Cycler Manual

Bio-Rad Laboratories Resources Bio-Rad Laboratories provides many resources for scientists. The following web sites contain useful information for running PCR and real-time PCR experiments: • Life Science Research web site (discover.bio-rad.com) This site includes links to technical notes, manuals, product information, and technical support. This site also provides many technical resources on a wide variety of methods and applications related to PCR, real-time PCR, and gene expression. Table 1 lists Bio-Rad Laboratories resources and how to locate what you need. Table 1. Bio-Rad resources. Resource

How to Contact

Local Bio-Rad Laboratories representatives

Find local information and contacts on the Bio-Rad Laboratories web site (www.bio-rad.com). Find the nearest international office in the list on the back of this manual. Technical notes and literature Go to the Bio-Rad Laboratories web site (www.biorad.com). Type a term in the Search box and select Documents to find links to technical notes, manuals, and other literature. Technical specialists To find local technical support on the phone, contact your nearest Bio-Rad Laboratories office. For technical support in the United States and Canada, call 1-800-424-6723 (toll-free phone), and select the technical support option.

Warranty The T100™ thermal cycler and associated accessories are covered by a standard Bio-Rad Laboratories warranty. Contact your local Bio-Rad Laboratories office for the details of the warranty.

Writing Conventions Used in This Manual This manual provides instructions on how to safely set up and operate the T100 thermal cycler and uses the writing conventions shown in Table 2 to quickly provide relevant information. Table 2. Manual convention. Convention

Meaning

TIP:

Provides helpful instructions, including information explained in further detail elsewhere in this manual Provides important information, including information explained in further detail elsewhere in this manual Explains crucial information about a topic that may lead to injury to the user, instrument damage, or data loss Touch X using your finger. For example, touch New Protocol means use your finger to touch the New Protocol button on the screen. From the menu X, touch Y. For example, touch Tools > About means touch the Tools button, then touch About from the Tools menu.

NOTE: WARNING! Touch X Touch X > Y

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Safety and Regulatory Compliance The T100 thermal cycler heats and cools very quickly during operation. We strongly recommend that you follow the safety specifications listed in this section and throughout this manual.

Safety Warning Labels Warning labels posted on the instrument and in this manual warn you about sources of injury or harm. Refer to Table 3 to review the meaning of each safety warning label. Table 3. Instrument safety warning labels. Icon

Meaning CAUTION: Risk of danger! This symbol identifies components that pose a risk of personal injury or damage to the instrument if improperly handled. Wherever this symbol appears, consult the manual for further information before proceeding. CAUTION: Risk of electrical shock! This symbol identifies components that pose a risk of electrical shock if improperly handled. CAUTION: Hot surface! This symbol identifies components that pose a risk of personal injury due to excessive heat if improperly handled.

Instrument Safety Warnings The following warning labels display on the instrument and refer directly to the safe use of this T100 thermal cycler (Table 4). Table 4. Instrument safety warning labels. Icon

Meaning Warning about risk of harm to body or equipment. Operating the T100 instrument before reading this manual can constitute a personal injury hazard. Only qualified laboratory personnel should operate this instrument. Warning about risk of harm to body or equipment from electrical shock. Do not attempt to repair or remove the outer case of this thermal cycler, power supply, heat pump, or other accessories. If you open these instruments, you put yourself at risk for electrical shock and void your warranty. All repairs must be done by an authorized repair service.

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Table 4. Instrument safety warning labels. (continued) Icon

Meaning Never remove the outer case of a thermal cycler base. This may cause you to receive an electrical shock. This thermal cycler uses neutral fusing, which means that live power could still be exposed inside the instrument even when the fuse is blown or removed. Warning about risk of burning. A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or other eye protection at all times during operation. Always allow the sample block to return to idle temperature before opening the lid and removing samples. Always allow maximum clearance to avoid accidental skin burns. Warning about risk of explosion. The sample blocks can become hot enough during the course of normal operation to cause liquids to boil and explode.

Safety and Regulatory Compliance This instrument has been tested and found to be in compliance with all applicable requirements of the following safety and electromagnetic standards (Table 5). Table 5. Safe use specifications. Usage Aspect

Conditions for Safe Use

Rated input power Overvoltage category Replacement fuses Environment Temperature Relative humidity Altitude Pollution degree

100–240 Vac, 50–60 Hz, 700 W Max II 10 A, 250 V, 5 x 20 mm, fast blow (qty 2) Indoor use only 15–31°C Up to 80% (noncondensing) Up to 2,000 meters above sea level 2

SAFETY COMPLIANCE This instrument has been tested and found to be in compliance with all applicable requirements of the following safety and electromagnetic standards: • IEC 61010-1:2001 Safety requirements for electrical equipment for measurement, control, and laboratory use -Part 1: General requirements • IEC 61010-2-010:2003 Safety requirements for electrical equipment for measurement, control, and laboratory use -Part 2-010: Particular requirements for laboratory equipment for the heating of material • CAN/CSA-C22.2 NO. 61010-1-04 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use - Part 1: General Requirements • CAN/CSA-C22.2 NO. 61010-2-010-04 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use - Part 2-010: Particular Requirements for Laboratory Equipment for the Heating of Materials • EN 61010-1:2001 Safety requirements for electrical equipment for measurement, control, and laboratory use -Part 1: General requirements

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• EN 61010-2-010:2003 Safety requirements for electrical equipment for measurement, control, and laboratory use -Part 2-010: Particular requirements for laboratory equipment for the heating of material • UL 61010-1:2004(R2008) Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use -Part 1: General Requirements • UL 61010A-2-010:2002 Standard for Electrical Equipment for Laboratory Use; Part 2: Particular Requirements for Laboratory Equipment for the Heating of Material

ELECTROMAGNETIC COMPATIBILITY (EMC) • IEC61326-1:2005 Electrical Equipment for measurement, control, and laboratory use -EMC Requirements, Class A • EN61326-1:2006 Electrical Equipment for measurement, control, and laboratory use -EMC Requirements, Class A • FCC Part 15, Subpart B, Sections 15.107 and 15.109 as a Class A digital device.

FCC WARNINGS AND NOTES WARNING! Warning: Changes or modifications to this unit, not expressly approved by Bio-Rad Laboratories, could void the user’s authority to operate the equipment. • Note: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference, at his own expense. • Note regarding FCC compliance: Although this instrument has been tested and found to comply with Part 15, Subpart B of the FCC Rules for a Class A digital device, please note that this compliance is voluntary, for the instrument qualifies as an “exempted device” under 47 CFR 15.103(c), in regard to the cited FCC regulations in effect at the time of manufacture. • Note regarding Canadian EMC compliance: Le present appareil numerique n’emet pas de bruits radioelectrique depassant les limites applicables aux appareils numeriques de class A prescrites dans le reglement sur le brouillage radioelectrique edicte par le Ministere des Communications du Canada.

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T100 Thermal Cycler Manual

Table of Contents Bio-Rad Laboratories Resources... iii Warranty... iii Writing Conventions Used in This Manual... iii Safety and Regulatory Compliance... iv

Chapter 1. Introduction to the T100™ Thermal Cycler... 1 System Overview... Setting Up the T100 Thermal Cycler... Home Screen Overview... Loading Samples... Compatible Reaction Vessels and Sealing Options...

1 2 3 3 4

Chapter 2. Creating, Editing, and Saving Protocols... 5 Creating a New Protocol or Editing an Existing Protocol... 5 Sample Volume and Lid Temperature... 11 Saving a Protocol... 12

Chapter 3. Running Protocols... 13 Starting a Run... Pausing and Resuming a Run... Skipping Steps in a Protocol... Canceling a Run... Incubating Samples...

13 15 16 16 16

Chapter 4. Managing Protocol Files and Folders... 17 Saved Protocols Overview... 17

Chapter 5. Tools... 19 Tools Menu... 19

Chapter 6. Maintenance and Cleaning... 23 Index... 27

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Introduction to the T100™ Thermal Cycler Read this chapter for information on setting up the T100 thermal cycler. • System overview (page 1) • Setting up the T100 thermal cycler (page 2) • Home screen (page 3) • Loading samples (page 3) • Selecting compatible reaction vessels and sealing options (page 4)

System Overview The T100 thermal cycler has a 96-well block that is compatible with standard height tubes, tube strips, and plates. It can run sample volumes of 1–100 µL, with volumes of 15–100 uL recommended for the best reproducibility. It is composed of the following parts: • Reaction block - holds the plates, tubes or tube strips containing samples • Heated inner lid - heats the top of the samples to prevent evaporation and condensation • Touch screen - provides the means to control the cycler with gloved or ungloved hands • USB port - connects to a USB flash drive for data transfer • Status LED - turns on during normal operation; blinks when the cycler is in standby • Air vents - provide ventilation to allow the thermal cycler to heat and cool quickly

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Heated inner lid

Reaction block

Status LED Touch screen

Air vents

USB port

Figure 1. Front view of the T100 thermal cycler.

Setting Up the T100 Thermal Cycler The T100 thermal cycler package includes: • T100 thermal cycler • Power cord • Support ring for individual tubes • Instruction manual Remove all packaging materials and store them for future use. If any item is missing or damaged, contact your local Bio-Rad office. Place the T100 thermal cycler base on a flat, dry surface with sufficient cool airflow to run properly. 1. Before operating the thermal cycler, be sure to read the safety specifications (Safety and Regulatory Compliance, on page iv) and operating requirements. 2. Plug the supplied power cord into the appropriate electrical outlet. 3. Turn on the thermal cycler using the power switch on the back panel. 4. The thermal cycler runs a self-test to verify proper function, and then displays the home screen.

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Home Screen Overview The home screen provides access to all the main functions of the thermal cycler (Figure 2), as well as the date, time, and name of the thermal cycler (optional).

Figure 2. The home screen of the T100 thermal cycler. To initiate the functions in the home screen, touch the button for each function: • New Protocol - creates a new protocol (page 5) • Saved Protocols - for viewing, editing, and running saved protocols (page 17) • Incubate - for running at a constant temperature similar to an incubator (page 16) • Tools - logs, settings, self-test, system information, and firmware update (page 19)

Loading Samples The inner lid of the reaction module applies heat and force to the reaction vessel lids (caps or sealing film). Heating the inner lid prevents condensation, and applying force seals the reaction to prevent evaporation. WARNING! After a run, the heated inner lid can remain hot. Use caution when opening and closing the lid. 1. To open the lid, lift up the lid handle until the lid stays open without assistance (Figure 3).

Figure 3. Lift the lid handle up to open the lid.

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2. Load the samples To ensure uniform heating and cooling of samples, vessels must be in complete contact with the reaction block. Adequate contact is achieved by: • Confirming that the block is clean before loading samples • Firmly pressing the individual tubes or the microplate into the block wells NOTE: When running individual tubes, place the support ring in the block to prevent deformation of the tubes by the heated lid. The ring is reversible for domed cap tubes or flat cap tubes (Figure 4). Alternatively, load eight empty tubes in the corners and sides of the block. 3. To close the lid, push the lid handle down until it stops.

Figure 4. Tube support ring for running a few individual tubes.

Compatible Reaction Vessels and Sealing Options The T100 thermal cycler is compatible with full-height strips, tubes, and plates. Bio-Rad tests the standard supplies listed in Table 1 for compatibility with the T100 thermal cycler. The composition and thickness of reaction vessels influence the outcome of a reaction. Microplates, tubes, sealers, and caps come in a variety of compositions and colors. For a full list of available reagents and supplies, refer to the Life Science Research product catalog or the website at discover.bio-rad.com. Table 1. Compatible microplates, tubes, and sealing options for the T100 thermal cycler. Tubes and Microplates Description (Catalog Prefix*)

Caps and Sealers Volume and Number

Description (Catalog Prefix*)

Tubes

Caps (TCS*)

Individual tubes with caps (TFI* and TWI*) 0.2 ml Strip tubes (TBS*) 0.2 ml, 8- and 12-tube Microplates Film (MS*)

(Included) Strip

Unskirted and full-height (MLP*) iQ full height plates (223-9441) Semi-skirted, full-height, and hard-shell (HSS*)

96-well 96-well 96- well

* The prefix of the catalog number for each series of supplies.

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A, B, F A, B, F A, B, F

Caps (TCS*) Strip Strip Strip

T100 Thermal Cycler Manual

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Creating, Editing, and Saving Protocols Read this chapter for information on creating, editing, and saving protocols. • Creating a new protocol or editing an existing protocol (page 5) • Sample volume and lid temperature (page 11) • Saving a protocol (page 12)

Creating a New Protocol or Editing an Existing Protocol To create or edit a protocol: 1. To create a new protocol, touch New Protocol from the home screen. 2. Alternatively, to edit an existing protocol, touch Saved Protocols from the home screen, select the protocol to be modified and touch Edit (Figure 5). 3. The protocol editing screen opens (Figure 6).

Figure 5. Select a protocol and touch Edit.

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Creating a New Protocol or Editing an Existing Protocol

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Figure 6. The template for a PCR protocol. 4. The protocol editor displays the protocol in a graphical format using editable boxes. 5. To edit the temperature or time for each step, the sample volume, and the lid temperature, touch the appropriate box and enter a new value in the numeric keypad. 6. Touch Run to run the protocol immediately without saving it. Alternatively, touch Save to choose a folder location and save the protocol, and then touch Run to run it (page 13).

Protocol Parameters Table 2 includes a list of all the parameters for temperature and gradient steps. Table 2. Protocol steps and parameters of the T100™ thermal cycler. Step Name

Parameters and Ranges

Temperature

Temperature in °C: The target Instructs the thermal cycler to ramp to temperature between 4.0 and the target temperature and hold that 100.0°C in tenths of a degree temperature for a specified time The hold time between 1 sec and 18 hr in the format of hr:min:sec. To enter an infinite hold, touch the ∝ (infinity, 0) key Instructs the thermal cycler to ramp to Front row: The lower the target temperature gradient across temperature in the gradient. the block and hold that temperature Enter a number between 30.0 and 99.0°C in tenths of a degree gradient for a specified time

Time

Gradient

Back row: The upper temperature in the gradient. The maximum temperature is 100°C. Enter a temperature within 25.0°C of the lower temperature

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Description

T100 Thermal Cycler Manual Table 2. Protocol steps and parameters of the T100™ thermal cycler. (continued) Step Name

Parameters and Ranges

Description

Increment

A temperature from –10.0 to 10.0°C per cycle in tenths of a degree

Ramp Rate

A number from 0.1 to 4°C per sec

Extend

A time from –60 to 60 sec per cycle

Applies only to a temperature step. Instructs the thermal cycler to increment (change) the target temperature of a step with each cycle, where a positive number increases the temperature and a negative number decreases the temperature Applies only to a temperature step. Instructs the thermal cycler to ramp to the target temperature at the specified ramp rate in that step. If not specified, the T100 thermal cycler runs at a maximum ramp rate of 4°C/sec Applies to both temperature and gradient steps. Instructs the thermal cycler to extend the hold time with each cycle. A positive number increases the hold time and a negative number decreases the hold time.

Changing Target Temperature and Hold Time To change the target temperature and hold time in a temperature step: 1. From the protocol editor, touch the temperature or hold time for a step. 2. A pop-up keypad appears to allow you to to edit the value (Figure 7). 3. Enter a new value and touch OK.

Figure 7. Editing a temperature with the numeric keypad.

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Creating a New Protocol or Editing an Existing Protocol

Inserting a Protocol Step Insert a protocol step if a new temperature, GOTO, or gradient step is needed: 1. Select a step to the left of where the new step is to be inserted. 2. Touch Insert. A pop-up menu displays types of steps to insert (Figure 8).

Figure 8. Insert a new step to the right of step 1. 3. Touch Temperature to insert a temperature step, Gradient to insert a gradient step, or GOTO to insert a GOTO step to create a cycle for PCR. 4. Edit the parameters in the new step as desired.

Deleting a Protocol Step To remove a step from a protocol: 1. Select the step to be deleted. 2. Touch Delete to remove the selected step.

Adding or Removing a Temperature Gradient To add a temperature gradient to a step: 1. Select a step and touch Options to open the Step Options window (Figure 9).

Figure 9. The Step Options window.

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2. Select the Gradient checkbox. The temperatures for each row are displayed (Figure 10). 3. Touch the Back Row and Front Row fields to edit the gradient, and touch OK. NOTE: A gradient range must be between 1 and 25°C. NOTE: A gradient step can also include an Extend parameter, but the Increment and Ramp Rate options are not allowed.

Figure 10. The Step Options window displays the gradient temperatures. 4. The front and back row temperatures of the gradient are displayed as two lines in the thermal profile (Figure 11). 5. The gradient can be edited by touching the temperatures directly without opening the Step Options screen.

Back and front

row temperatures

Figure 11. The protocol has a gradient from 62 to 72°C. To remove the temperature gradient: 1. Select the gradient step and touch Options to open the Step Options window. 2. Deselect the Gradient checkbox. 3. Select OK to confirm.

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Creating a New Protocol or Editing an Existing Protocol

Temperature Increment, Extend, and Ramp Rate To add increments, extensions, and ramp rates to a step: 1. Select a step and touch Options to open the Step Options window (Figure 12). 2. A step can contain all three options. To edit an option, touch the field for Increment, Extend, or Ramp rate. 3. Enter a value using the numeric keypad, and touch OK.

Figure 12. Step 4 has a temperature increment option. To remove the increment, extension, or ramp rate, 1. From the Step Options window, touch the field for the option and change the value to zero using the numeric keypad. 2. Touch OK to confirm.

Changing Parameters in a GOTO Step The GOTO step instructs the thermal cycler to repeat a set of steps in a cycle. To edit the GOTO step: 1. Select a GOTO step.

Figure 13. A protocol that includes a GOTO step 2 and 34 additional repeats. 2. To change the step that the cycler will start repeating, touch the Step field.

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3. Enter a value for the first step in the GOTO loop using the numeric keypad and touch OK. 4. To change the number of times the cycler will repeat the cycle, touch the X field. 5. Edit the number of repeats using the numeric keypad and touch OK. NOTE: The X field is the number of additional repeats. For example, 34 X will repeat steps 2–4 an additional 34 times for a total of 35 cycles of PCR. NOTE: A protocol may contain multiple GOTO loops but they may not be nested with one loop inside another.

Sample Volume and Lid Temperature • Sample volume - determines the temperature control mode, which influences the amount of time the samples are held at the target temperature • Lid temperature - determines the temperature of the heated lid The T100 thermal cycler provides three ways to enter sample volume and lid temperature: • Change the setting when creating and editing the protocol (page 12) • Override the sample volume setting while initiating a run (page 14) • Set a default for the sample volume for new protocols in Settings (page 20)

Temperature Control Modes The T100 thermal cycler uses one of two temperature control modes to determine when the sample reaches the target temperature: • Calculated mode - the thermal cycler calculates the sample temperature based on the sample volume when a sample volume between 1 and 100 µl is entered. • Block mode - when a sample volume of zero (0) µl is entered, the thermal cycler assumes that the sample temperature is the same as the measured block temperature

Changing Sample Volume To change the sample volume for a protocol: 1. From the New Protocol screen, touch the Volume field. 2. Using the numeric keypad, enter the sample volume and touch OK. 3. Entering a sample volume between 1 and 100 µl selects Calculated mode. Entering a sample volume of zero (0) selects Block mode. Calculated mode is the recommended mode because it most accurately represents the actual sample temperature.

Changing Lid Temperature Heating the lid prevents condensation from forming inside the reaction wells. Without a heated lid, water can be lost to condensation, concentrating the reactants in the tube or plate. The T100 thermal cycler lid can be programmed to heat between 40 and 110°C. The default recommended lid temperature is 105°C. NOTE: When the block is running an infinite hold at a temperature below the cutoff temperature of 30.0°C, the lid heater maintains 31.0°C.

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