User Bulletin. Applied Biosystems StepOne Real-Time PCR System DRAFT. SUBJECT: StepOne System Updates. In This Bulletin Installation Updates...

Transcription

1 Applied Biosystems StepOne Real-Time PCR System User Bulletin User Bulletin Applied Biosystems StepOne Real-Time PCR System January 2007 SUBJECT: StepOne System Updates In This Bulletin Installation Updates Networking Updates Maintenance Updates Documentation Updates Getting Started Guides Installation Guide How to Obtain Support Overview Visit the StepOne System Website This bulletin contains important updated information for installing, maintaining, and networking the Applied Biosystems StepOne Real-Time PCR System. If you are responsible for installing or maintaining the StepOne system, read this bulletin before using the instrument. Applied Biosystems now offers a website devoted to the StepOne system. For the latest services and support information for the StepOne instrument, go to: info.appliedbiosystems.com/stepone The website contains the latest software and documentation updates for the StepOne instrument and analysis software. At the StepOne system support page, you can: Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download documents in Adobe portable document format (PDF) Obtain information about customer training Download software updates and patches for the StepOne instrument

2 StepOne System Updates Installation Updates Read this section before installing the StepOne instrument. Additional Guidelines and Best Practices Vibration and Instrument Placement Avoid placing the StepOne instrument on surfaces that are subject to constant or intermittent vibration. Tabletop centrifuges, vortex mixers, and other laboratory equipment can vibrate the instrument during a run and produce data collection errors. Packing Material Removal When removing the packing material from the sample block, you must power on the StepOne instrument to lower the instrument drawer. The drawer is in the raised position during transportation and it will not open until the instrument is powered on. Instrument Boot Time When you power on the instrument for the first time, the instrument may require more than five minutes to boot. Proceed with the installation when the touchscreen displays the main screen, indicating that the StepOne instrument has completed the boot. Note: If you are updating the StepOne instrument firmware, the instrument may pause for up to 20 min before completing the boot. PCMCIA Card Installation If you ordered a laptop computer with your StepOne instrument, wait several minutes after installing the PCMCIA network card to allow the computer to enable the device. The computer may require time to initialize the drivers and register the network card with the operating system before it is available for use. Note: If you are installing your StepOne system in a colocated setup, do not connect the StepOne system cable to the PCMCIA card Ethernet port. If you have already installed the instrument connection through the card, unplug and connect the StepOne system cable to the appropriate port. 2 Applied Biosystems StepOne Real-Time PCR System User Bulletin

3 Networking Updates Networking Updates Read this section before connecting the StepOne system to a network. Additional Guidelines and Best Practices Consult a Network Administrator Applied Biosystems recommends that you consult a network administrator before connecting the StepOne system to your laboratory network. Confirm the Instrument Connections Confirm that the StepOne instrument is connected to the network by the blue LAN Port ( ). If the StepOne system is installed in a colocated layout, confirm that the instrument is connected to the colocated computer by the yellow Computer Port ( ). Limit Remote Monitoring to One Computer Avoid using more than one computer simultaneously to remote monitor the instrument. Although the StepOne system supports remote monitoring from multiple computers, each connection taxes the instrument microprocessor. Too many connections can overburden the StepOne instrument and result in instrument errors. Note: The effects of an overburdened StepOne instrument are evident in the Temperature Plot during a run. Symptoms can include extended hold times or brief, unexpected plateaus in the instrument temperature plot. Note Restrictions to mdns and Auto Discovery The StepOne instrument supports multicast domain name service (mdns) but only when the instrument and computer share a direct network connection and are within the same subnet. Consequently, computers on the network that are separated from the StepOne instrument by a router, hub, or other network device may not be able to access the instrument by its host name. The StepOne instrument also does not support auto discovery for mdns. However, the instrument does support name resolution, but the instrument name must be unique within the subnet. Note: Confirm the uniqueness of the instrument name manually. The StepOne instrument does not test the uniqueness of the instrument name when it is set. Add Instruments Using Lower Case Letters When you add the StepOne instrument for remote monitoring, enter the instrument name using lower case letters only. Applied Biosystems StepOne Real-Time PCR System User Bulletin 3

4 StepOne System Updates Troubleshooting Workflow Use the following workflow to troubleshoot the network connection to the StepOne system when setting up the instrument for remote monitoring. Start StepOne software cannot add instrument for remote monitoring Is the instrument connected to the network by the blue LAN Port? No Yes Determine the IP address and network settings of the instrument LAN port as explained in step 1 on page 5. Is the LAN Port of the instrument set up for static IP or DHCP? DHCP Static IP Determine the subnet address for the instrument as explained in How to Determine the Subnet Address on page 6. Connect the Ethernet cable for the network connection to the blue LAN Port at rear of the StepOne instrument. Confirm with your network administrator that the static IP Address, Subnet Mask, and Default Gateway settings are correct. Correct the network settings as directed by your network administrator. Are the instrument and computer located on the same subnet? No Add and monitor the instrument using the IP address of the LAN Port. Yes Confirm the network connection by pinging the instrument as explained in How to Ping the Instrument on page 5. Can you ping the instrument from a computer on the network? No Consult your network administrator to determine the source of the failed network connection. Yes Can you add or monitor the instrument using the StepOne software? Contact Applied Biosystems as explained in How to Obtain Support on page 11. No When you add the instrument for remote monitoring, enter the instrument name using lower case letters only. If you cannot use the instrument name to add the instrument for remote monitoring, try using the IP address of the instrument. Unplug the instrument from the network, then ping the IP address. If you can ping the address after removing the instrument from the network, then another device is using the same IP address and you must reassign it to resolve the conflict. 4 Applied Biosystems StepOne Real-Time PCR System User Bulletin

5 Networking Updates How to Ping the Instrument Note: The procedure below is a revision of the Confirm the Network Connection procedure found in the Applied Biosystems StepOne Real-Time PCR System Installation Guide. You can confirm the connection between a network computer and StepOne instrument by pinging the instrument name or IP address from the computer. The ping command issued from the DOS command shell causes the computer to send a series of 32-byte data packets to the instrument. If the network connection between the computer and the StepOne instrument is unobstructed, the instrument sends data packets in reply. 1. Determine the IP address of the StepOne instrument: a. Touch the StepOne instrument touchscreen to awaken it, then touch. b. In the Main Menu, touch Settings Menu Admin Menu Set IP Address. c. Record the IP address in the IP Address field. 2. Ping the StepOne instrument: a. Power on the StepOne instrument and wait for the instrument to boot. b. In the computer desktop, select Start Programs Accessories Command Prompt. c. In the Command Prompt window, enter ping <IP address>, then press Enter. If the DOS prompt displays replies to the pings request timed out Then the computer can communicate with the IP address. Wait a few minutes for the network to update the IP address of the instrument, then ping the instrument again. If you still cannot ping the StepOne instrument after two attempts, ping the IP address of the alternate port. Consult your network administrator to determine the source of the failed network connection. Resolving an IP Address Conflict If you suspect that the IP address used by either port of the StepOne instrument may conflict with another device on your network, you can use the following procedure to test for the conflict. 1. Unplug the instrument from the network. 2. Ping the IP address of the instrument LAN port as explained above. If you can ping the IP address, then another device on the network is already using the address and you must reassign the IP address of the instrument to resolve the conflict. Applied Biosystems StepOne Real-Time PCR System User Bulletin 5

6 StepOne System Updates How to Determine the Subnet Address 1. Touch the StepOne instrument touchscreen to awaken it, then touch. 2. In the Main Menu, touch Settings Menu Admin Menu Set IPAddress. 3. Record the IP address in the IP Address field. The first three numbers of the IP address are the subnet address. For example, the italicized numbers in the following IP address are the subnet address: If the subnet address for the StepOne instrument is different than that of the computer used for remote monitoring, then you cannot use the instrument name to add the instrument for remote monitoring. The mdns limitation explained in Additional Guidelines and Best Practices on page 3 restricts the use of the instrument name as an identifier to network devices on the same subnet. Note: To determine the subnet address of the computer that you want to use to monitor the StepOne instrument, select Start Help and Support in the computer desktop, then use the Microsoft Windows Help System search for the procedure appropriate for the operating system. Maintenance Updates Read this section before maintaining the StepOne instrument. Guidelines for Bleach Solution Use Applied Biosystems recommends the use of 10% bleach solution for resolving fluorescent contamination on the StepOne instrument sample block. However, excessive use of the solution can corrode the block material. To prevent degradation of the block, observe the following guidelines when using bleach solution: Decontaminate the sample block using bleach solution as a last resort. Rinse the sample block with bleach only after treatments of deionized water and 95% ethanol fail to remove the contamination. Rinse the sample block thoroughly using deionized water after treating the block with bleach solution. Thoroughly removing residual bleach from the metal surfaces with water will minimize the long term effects of bleach. 6 Applied Biosystems StepOne Real-Time PCR System User Bulletin

7 Documentation Updates Documentation Updates Getting Started Guides This addendum to the StepOne system documentation describes the latest updates to the StepOne software and StepOne instrument touchscreen user interface. The updates below apply to the following StepOne system getting started guides: Applied Biosystems StepOne Real-Time PCR System Getting Started Guide for Genotyping Experiments Applied Biosystems StepOne Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C T ( C T ) Experiments Applied Biosystems StepOne Real-Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1: Get Started About Data Collection The StepOne system can collect real-time data for presence/absence experiments. Advantages of TaqMan Reagents With the design wizard, the presence/absence experiments use TaqMan reagents in the PCR reactions. If you want to perform a presence/absence experiment using other reagents to detect amplification of the target sequence, set up an experiment using advanced setup. With the design wizard, the genotyping and presence/absence experiments use the standard ramp speed in the instrument run. If you want to perform a genotyping or presence/absence experiment using the Fast ramp speed, set up the experiment using advanced setup. IMPORTANT! Applied Biosystems does not support the use of SYBR Green or TaqMan Fast reagents for presence/absence or genotyping experiments. Note: The Reaction Setup screen is not available if you are using other reagents. This note is to clarify the design wizard setup for quantitation experiments. Chapter 2: Design the Experiment Set Up the Targets (Quantitative PCR Only) When you use the Design Wizard to set up a quantitation experiment (standard curve, relative standard curve, or comparative C T ), you can set up only singleplex reactions (amplification and detection of one target sequence). To set up a quantitation experiment with multiplex reactions (amplification and detection of two target sequences), set up the experiment using Advanced Setup. Note: For more information on Advanced Setup, access the Help from within the StepOne software by pressing F1, click in the toolbar, or selecting Help StepOne Help. Applied Biosystems StepOne Real-Time PCR System User Bulletin 7

8 StepOne System Updates Chapter 4: Run the Experiment About the Run Method Screen The Run Method screen is not updated throughout the run; however, the Run Status element of the Amplification, Temperature, and Run Method screens are updated continuously. Note: The Run Method screen does not behave similarly to the standalone monitoring that appears in the StepOne instrument interface. Colocated Data Transfer The last line of the paragraph should read: The StepOne software automatically transfers the experiment data from the StepOne instrument after the run. Load the Instrument 2. Place the reactions in the sample block: If using a reaction plate: Place the reaction plate in the sample block with well A1 at the back-left corner. If using reaction tube strips: Place the tray containing the tube strips in the sample block. If using reaction tubes: Place the tray containing the tubes in the sample block. IMPORTANT! Load at least 4 tubes for optimal performance. Chapter 5: Analyze the Experiment View the Amplification Plot The background is not a characteristic of the amplification curve as are the baseline, exponential (geometric phase), linear, and plateau phases. Background describes the noise that remains after the StepOne software compensates for the baseline, which is present throughout the amplification curve, not just in one region. The other four phases describe distinct sections of the Amplification curve. View the Multicomponent Plot The Multicomponent Plot can display the data for multiple wells simultaneously. Appendix A View the Raw Data Plot The legend of the Raw Data Plot displays the color coding for each row of the plate. Export/Import Workflow When importing setup data, it is easier to set up a new experiment using the data exported from a similar experiment. 8 Applied Biosystems StepOne Real-Time PCR System User Bulletin

9 Documentation Updates Glossary baseline-corrected normalized reporter ( Rn) / delta Rn ( Rn) For presence/absence experiments, the difference in the normalized reporter value (R n ) between the pre-read and the post-read of the data calculated as follows: Installation Guide IPC+ R n = R n (Post Read) R n (Pre Read) No longer used. See negative control - IPC wells. threshold [1] Level of fluorescence above the baseline and within the exponential growth region of the amplification plot. The threshold can be determined automatically (see automatic C T ) or can be set manually (see manual C T ). [2] Level of fluorescence above which the StepOne software assigns a positive call to a well. The updates below apply to the Applied Biosystems StepOne Real-Time PCR System Installation, Network, and Maintenance Installation Guide. Chapter 1: Get Started Minimum Computer Requirements If you did not purchase a computer with your StepOne system, supply one that meets the following requirements: Component Minimum Requirements Computer Intel processor, 1.0 GHz 512 MB of RAM One 20-GB hard drive 20/48X IDE CD-ROM Ethernet network interface adapter (10BASE-T) UL-listed CE-marked FCC-labelled Monitor pixel resolution 32-bit color UL-listed Operating System Microsoft Windows XP Professional Operating System, Service Pack 2 or higher A computer that meets the minimum requirement provides optimal software performance and is supported by Applied Biosystems. Necessary only if you plan to connect the computer to the StepOne instrument or to a local area network. Applied Biosystems StepOne Real-Time PCR System User Bulletin 9

10 StepOne System Updates Chapter 2: Set Up the StepOne Instrument Define the Settings Reverse the order of steps for changing the instrument date and time. Also, note that the Touchscreen Calibration is no longer part of the installation. Instrument Standby When the power button ( ) is touched, the StepOne instrument interface displays a Confirmation Screen that prompts you to enter Standby mode. Touch Yes to enter standby mode. Chapter 5: Connect the StepOne System to a Network Chapter 6: Maintain the StepOne System Appendix A: How to Troubleshoot the Installation Define the LAN Port IP Settings Step 2 should read: In the Main Menu, touch Settings Menu, touch Admin Menu, then touch Set IP Address. (Optional) Define the Computer Port IP Settings The StepOne instrument no longer permits changes to the IP settings of the Computer Port. The IP address settings accessed from the Set IP Address command of the Admin Menu are for the LAN Port. Decontaminate the Sample Block Note the importance of rinsing the sample block using deionized water following treatment with bleach solution as explained in Guidelines for Bleach Solution Use on page 6 of this bulletin. Network Connection Problems Add the Troubleshooting Workflow on page 4 of this bulletin to the section to aid in troubleshooting failed network connections when setting the StepOne instrument for remote monitoring. Confirm the Network Connection Replace with the procedures in How to Ping the Instrument on page 5 of this bulletin. 10 Applied Biosystems StepOne Real-Time PCR System User Bulletin

11 How to Obtain Support How to Obtain Support For the latest services and support information for all locations, go to then click the link for Support. At the Support page, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. Applied Biosystems Customer Care Center Contact the Applied Biosystems Customer Care Center when directed by this guide, or to schedule maintenance for your StepOne instrument (such as, annual planned maintenance or temperature verification/calibration). To contact the Applied Biosystems Customer Care Center call , Option 1 (U.S.A. only). For all other countries, please contact your local Applied Biosystems service representative. Applied Biosystems StepOne Real-Time PCR System User Bulletin 11

12 Copyright 2007, 2010 Applied Biosystems. All rights reserved. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. NOTICE TO PURCHASER: Label License The Applied Biosystems StepOne Real-Time PCR System is covered by US patents and corresponding claims in their non-us counterparts, owned by Applied Biosystems. No right is conveyed expressly, by implication, or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5 nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: Applera, Applied Biosystems, and AB (Design) are registered trademarks, and StepOne is a trademark of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at Applied Biosystems is committed to providing the world s leading technology and information for life scientists. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: /2010 Copyright / Back Page Part Number Rev. B