User Bulletin. ABI PRISM 310 Genetic Analyzer. A Fast Native Protocol for the Analysis of PCR Products. In This Bulletin

Transcription

1 User Bulletin June 23, 1999 (updated 12/2000) A Fast Native Protocol for the Analysis of PCR Products In This Bulletin This User Bulletin describes the Fast Native Protocol for the separation of DNA fragments that are 500 bp in length. The following topics are covered in this User Bulletin: Topic See Page Safety 2 Before You Begin 3 Material Preparation 4 Preparing for a Run 6 Analyzing the Data 9 Technical Support 12 What is the Fast Native Protocol? Advantages Limitations The Fast Native Protocol is a rapid capillary electrophoresis protocol for separating DNA fragments that are 500 bp in length. The Fast Native Protocol is run on the using a shortened (41-cm) capillary and 7-minute analysis times.the sizing resolution is at least 5 bp. The advantages of the Fast Native Protocol are as follows: Separates DNA fragments using 7-minute analysis times Processes 96 samples in less than 12 hours Assesses the performance of specific reactions PCR, for sizing of fragments that differ by at least 5 bp Minisequencing Detection of the absence or presence of DNA fragments in a sample The Fast Native Protocol has only been shown to separate DNA fragments that are 500 bp in length.

2 Safety Documentation User Attention Words Five user attention words appear in the text of all Applied Biosystems user documentation. Each word implies a particular level of observation or action as follows. Note This word is used to call attention to information. IMPORTANT This word calls attention to information that is necessary for correct operation of the kit or instrument. CAUTION This word informs the user that damage to the instrument could occur if the user does not comply with the information. It also indicates a potentially hazardous situation that could result in minor or moderate injury to the user.! WARNING! This word informs the user that serious physical injury to the user or other persons could occur if these required precautions are not taken. DANGER Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. General Warning! WARNING! CHEMICAL HAZARD. Some chemicals used with Applied Biosystems instruments or kits may be hazardous and can cause injury, illness, or death. Check regularly for chemical leaks and chemical spills, which represent potential safety hazards to the operator. If a leak or a spill occurs, follow clean-up instructions in the Material Safety Data Sheets (MSDSs), or in the Waste Profile in the Site Preparation and Safety Guide. Hazardous Chemical Warnings are prominently displayed on the labels of all hazardous materials. Always read the appropriate MSDSs and Waste Profiles before using the chemicals in any way. User Safety Precautions Ordering MSDSs Always wear the appropriate protective gloves, clothing, and eyewear when handling chemicals. Material Safety Data Sheets (MSDSs) for hazardous chemicals manufactured by Applied Biosystems will accompany your first shipment. To receive additional copies of MSDSs at no extra cost, call Applied Biosystems at (800) For chemicals required for this instrument but not manufactured or sold by Applied Biosystems, please obtain the MSDSs from their manufacturers. Ordering Kits and Reagents To order ABI PRISM kits or reagents, please contact Applied Biosystems at one of the regional sales offices listed in the Technical Support section at the end of this User Bulletin. Please have the part number of the kit or reagent you are ordering available when ordering. Page 2 of 16

3 Before You Begin Materials Required x You will need the following materials to perform a Fast Native Protocol run: Equipment/Reagent Item ABI PRISM 310 Data Collection Software, version 1.2 or higher GeneScan Analysis Software for the 310 Genetic Analyzer, version or higher Glass syringe, 1.0-mL GeneScan capillary, 47-cm x 50-mm i.d. (labeled with a green mark) Genetic Analyzer capillary cutters Source See your local Applied Biosystems representative Applied Biosystems (P/N ) Applied Biosystems (P/N ) Applied Biosystems (P/N ) 310 GeneScan Polymer (7%) Applied Biosystems (P/N ) 310 Genetic Analyzer Buffer with EDTA Applied Biosystems (P/N ) Dye Primer Matrix Standards Kit Fluorescent Amidite Matrix Standards Kit plus NED Matrix Standard GeneScan -350 Internal Lane Size Standard GeneScan -500 Internal Lane Size Standard Genetic Analyzer Buffer Vials (4.0-mL) or or Applied Biosystems (P/N ) Applied Biosystems (P/N ) (P/N ) Applied Biosystems (P/N ) (P/N ) Applied Biosystems (P/N ) ABI PRISM 310 Genetic Analyzer User s Manual (P/N ) GeneScan Analysis Software User s Manual (P/N ) GeneScan Reference Guide (P/N ) 1.5-mL Eppendorf tube with the lid removed Major Laboratory Suppliers (MLS) Magnifying glass MLS Metric ruler MLS Heat block MLS Deionized formamide MLS Note Use high-quality deionized formamide. Page 3 of 16

4 Material Preparation Shortening the Capillary To cut the capillary: Step Action 1 Have one of each at hand: GeneScan capillary (green) Capillary cutter Metric ruler Magnifying glass 2 Align the 41-cm end of the capillary on top of the metric ruler. 3 Using the capillary cutter, score the capillary at 6 cm from the end farthest from the capillary window. 4 Break off the capillary at the score. The total capillary length is now 41 cm. Note Properly dispose of the 6-cm piece of the capillary. 5 Check for a clean cut at the tip with a magnifying glass. If the cut does not appear clean, smooth the end by lightly brushing the flat end of the capillary cutter over the end of the capillary. Preparing Stock Solutions Prepare stocks of the following reagents/buffers to save time during run setup: Fast Native Protocol buffer (0.6X 310 Genetic Analyzer Buffer with EDTA) Fast Native Protocol polymer (2% 310 GeneScan Polymer solution in 0.6X 310 Genetic Analyzer Buffer with EDTA) Note Use the Fast Native Protocol buffer and polymer in place of the standard solutions when running samples. Making the Fast Native Protocol Buffer Note The Fast Native Protocol buffer lasts for 4 weeks at 2 6 C. Warm the buffer to room temperature before use. To prepare 50 ml of Fast Native Protocol buffer: Step Action 1 To a 50-mL polypropylene tube add: 3.0 ml 310 Genetic Analyzer Buffer with EDTA 47 ml deionized water 2 Mix thoroughly. Page 4 of 16

5 Making the Fast Native Protocol Polymer Note Fast Native Protocol polymer lasts for 4 weeks at room temperature. To prepare 50 ml of Fast Native Protocol polymer: Step Action 1 To a 50-mL polypropylene tube add: 14.3 ml 310 GeneScan Polymer (7%) 3.0 ml 310 Genetic Analyzer Buffer with EDTA 32.7 ml deionized water 2 Mix thoroughly. Page 5 of 16

6 Preparing for a Run Instrument Setup Setting the Run Temperature At Any Time Before a Run Preparing and Loading Samples Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for the general procedure. Set the instrument run temperature to 60 C immediately before run setup. Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for details. Perform the following at any time before beginning a run: Complete the Sample Sheet. Refer to the ABI PRISM GeneScan Analysis Software Manual for details. Create an appropriate Fast Native Protocol matrix file for the chosen dye set. For directions on preparing matrix samples for Fast Native Protocol analysis, see Preparing the Matrix Samples on page 7. Create and then reuse a single matrix file for each set of run conditions. For DNA fragments that are considerably smaller than 500 bp, determine in a trial run the approximate electrophoresis time in seconds. Then replace the default run time with this sample-specific run time. To prepare and load the samples: Step Action 1 Label the sample tubes with a permanent marker. 2 To each tube add: 10.5 µl deionized formamide 1 µl GeneScan Internal Lane Size Standard (GeneScan-350 or GeneScan-500 recommended) 1 µl sample Note When analyzing DNA fragments that are considerably smaller than 500 bp, determine in a trial run the approximate electrophoresis time in seconds. Then replace the default run time with this sample-specific run time.! WARNING! CHEMICAL HAZARD. Formamide is a known teratogen (i.e., it can cause birth defects). Wash thoroughly after handling formamide. Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of the MSDS from the manufacturer. 3 Place the samples in a sample tray. 4 Place the samples in a heat block or thermal cycler for 5 minutes at 95 C. 5 Remove the samples from the heat source and place immediately in an ice-water bath. 6 Place the sample tray on the autosampler. Note The 96-well tray used in the GeneAmp PCR System 9700 requires a tray adaptor (P/N ) to be used with the ABI PRISM 310 autosampler. Page 6 of 16

7 (ll Preparing the Matrix Samples To prepare the matrix samples: Step Action 1 To each of four sample tubes add: 11 µl deionized formamide 1 µl each of one of the matrix standards! WARNING! CHEMICAL HAZARD. Formamide is a known teratogen (i.e., it can cause birth defects). Wash thoroughly after handling formamide. Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of the MSDS from the manufacturer. 2 Place the standards in a heat block or thermal cycler for 5 minutes at 95 C. 3 Remove the standards from the heat source and place immediately in an ice-water bath. 4 Run these four injections either alone or in the first four injections of an experimental run. Run Modules The Fast Native Protocol has two dedicated modules: Fast GS Template A and Fast GS Template D. The default values for the Fast Native Protocol modules are: Module Value Run Time 300 seconds Electrophoresis Voltage 15 kv Electrophoresis Temperature 60 C Injection Time 5 seconds Injection Voltage 15 kv Starting the Run To start the run: Step Action 1 Install a new, 41-cm capillary as described in Shortening the Capillary on page 4. 2 Select Change Capillary from the Instrument window. Then click OK in the Reset window to reset the injection counter. 3 Set up the Sample Sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual. Note Prepare the Sample Sheet at any time prior to the run and save it to the Sample Sheet folder. 4 Select New from the File pull-down menu and click the GeneScan Injection List icon. Page 7 of 16

8 To start the run: (continued) Step Action 5 Set up the Injection List as described in the ABI PRISM 310 Genetic Analyzer User s Manual. Be sure to do the following: Select the appropriate Sample Sheet from the Sample Sheet pop-up menu. Select module Fast GS Template A or Fast GS Template D from the Module pop-up menu for every injection. IMPORTANT The displayed value in Run Time is in seconds, not minutes. To change the default value, calculate accordingly. 6 Either: Select the appropriate matrix file from the Matrix pop-up menu for every injection. or Start the run and assign the matrix to the sample files later. Note If you have not already created a matrix file, see Preparing the Matrix Samples on page 7. Refer to Appendix B of the GeneScan Reference Guide for instruction on creating a matrix file. 7 Click the Start button. Note If you have not preheated the heat plate, the module has an initial step in which the plate is heated to the selected run temperature before the first sample is injected. Page 8 of 16

9 Analyzing the Data Creating a Matrix File You must create a matrix file before analyzing the Fast Native Protocol data for the first time. For more information on creating matrices, refer to Appendix B of the GeneScan Reference Guide. For directions on preparing matrix samples for Fast Native Protocol analysis, see Preparing the Matrix Samples on page 7. Be sure to denature the matrix samples before loading. To create the matrix file, select the matrix standards and module as shown below. If using... Use module... And name it... Dye Primer Matrix Fast GS Template A Fast PCR Matrix A Standards Kit Fluorescent Amidite Matrix Standards Kit plus NED Matrix Standard Fast GS Template D Fast PCR Matrix D Note Usually, you create and save a single matrix file for each set of run conditions. However, if you experience persistent problems, such as spectral overlap in the analyzed data, you should remake the matrix file even if you have not altered the run conditions. Page 9 of 16

10 Setting the Analysis Parameters IMPORTANT If you choose to use different analysis parameters from those recommended here, be sure to define the Analysis Range to exclude the primer peak. To set the analysis parameters: Step Action 1 Open the GeneScan Analysis Software (version or higher). 2 Choose New from the File menu and click the Analysis Parameters icon. 3 Specify the settings as shown below. Note When analyzing PCR products, in Analysis Range choose a value for the start position that begins just after the primer peak. 4 Choose Save As... from the File menu. 5 Save the settings as Fast Native Analysis Parameters. 6 Click OK when done. Defining the Size Standard Define the size standard so that each unknown peak is flanked by at least two size standard peaks on either side. Refer to the GeneScan Analysis Software User s Manual for detailed protocols. Page 10 of 16

11 Internal Size Standard Shown below is the electropherogram for the GeneScan-500 internal lane size standard, labeled with ROX. Peaks representing 100, 200, 300, 400, and 500 bp are shaded. The table indicates the electrophoresis time (in minutes) for these peaks. Note When assigning the size values to the internal size standard peaks, do not assign a value to the 250-bp peak, since this fragment shows an anomalous migration behavior under the Fast Native Protocol conditions. Page 11 of 16

12 Technical Support Contacting Technical Support To Contact Technical Support by You can contact Applied Biosystems for technical support by telephone or fax, by , or through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems Web site (please see the section To Obtain Documents on Demand following the telephone information below). Contact technical support by for help in the following product areas: Product Area Genetic Analysis (DNA Sequencing) Sequence Detection Systems and PCR Protein Sequencing, Peptide and DNA Synthesis Biochromatography, PerSeptive DNA, PNA and Peptide Synthesis systems, CytoFluor, FMAT, Voyager, and Mariner Mass Spectrometers Applied Biosystems/MDS Sciex Chemiluminescence (Tropix) address galab@appliedbiosystems.com pcrlab@appliedbiosystems.com corelab@appliedbiosystems.com tsupport@appliedbiosystems.com api3-support@sciex.com tropix@appliedbiosystems.com Hours for Telephone Technical Support In the United States and Canada, technical support is available at the following times: Product Hours Chemiluminescence 8:30 a.m. to 5:30 p.m. Eastern Time Framingham support 8:00 a.m. to 6:00 p.m. Eastern Time All Other Products 5:30 a.m. to 5:00 p.m. Pacific Time To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below. (To open a service call for other support needs, or in case of an emergency, dial and press 1.) Product or Product Area Telephone Dial... ABI PRISM 3700 DNA Analyzer , then press 8 DNA Synthesis , then press 21 Fluorescent DNA Sequencing , then press 22 Fluorescent Fragment Analysis (includes GeneScan applications) Integrated Thermal Cyclers (ABI PRISM 877 and Catalyst 800 instruments) , then press , then press 24 Fax Dial Page 12 of 16

13 Product or Product Area ABI PRISM 3100 Genetic Analyzer , then press 26 BioInformatics (includes BioLIMS, BioMerge, and SQL GT applications) Peptide Synthesis (433 and 43X Systems) Protein Sequencing (Procise Protein Sequencing Systems) Outside North America , then press , then press , then press 32 PCR and Sequence Detection , then press 1 for PCR, 2 for the 7700 or 5700, 6 for the 6700 or dial , then press 5 Voyager MALDI-TOF Biospectrometry and Mariner ESI-TOF Mass Spectrometry Workstations Biochromatography (BioCAD Workstations and Poros Perfusion Chromatography Products) Expedite Nucleic acid Synthesis Systems Peptide Synthesis (Pioneer and 9050 Plus Peptide Synthesizers) , then press , then press , then press , then press 15 PNA Custom and Synthesis , then press 15 FMAT 8100 HTS System and , Cytofluor 4000 Fluorescence Plate then press 16 Reader Chemiluminescence (Tropix) Telephone Dial (U.S. only), or Applied Biosystems/MDS Sciex Telephone Region Dial... Africa and the Middle East Africa (English Speaking) and West Asia (Fairlands, South Africa) Fax Dial... Fax Dial South Africa (Johannesburg) Middle Eastern Countries and North Africa (Monza, Italia) 39 (0) (0) Eastern Asia, China, Oceania Australia (Scoresby, Victoria) China (Beijing) Page 13 of 16

14 Region Telephone Dial... Fax Dial... Hong Kong Korea (Seoul) / Malaysia (Petaling Jaya) Singapore Taiwan (Taipei Hsien) Thailand (Bangkok) Europe Austria (Wien) 43 (0) (0) Belgium 32 (0) (0) Czech Republic and Slovakia (Praha) Denmark (Naerum) Finland (Espoo) 358 (0) (0) France (Paris) 33 (0) (0) Germany (Weiterstadt) 49 (0) (0) Hungary (Budapest) 36 (0) (0) Italy (Milano) 39 (0) (0) Norway (Oslo) Poland, Lithuania, Latvia, and Estonia 48 (22) (22) (Warszawa) Portugal (Lisboa) 351 (0) (0) Russia (Moskva) South East Europe (Zagreb, Croatia) Spain (Tres Cantos) 34 (0) (0) Sweden (Stockholm) 46 (0) (0) Switzerland (Rotkreuz) 41 (0) (0) The Netherlands (Nieuwerkerk a/d 31 (0) (0) IJssel) United Kingdom (Warrington, Cheshire) 44 (0) (0) All other countries not listed 44 (0) (0) (Warrington, UK) Japan Japan (Hacchobori, Chuo-Ku, Tokyo) Latin America Del.A. Obregon, Mexico Page 14 of 16

15 To Reach Technical Support Through the Internet We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products. You can also order technical documents or an index of available documents and have them faxed or ed to you through our site. The Applied Biosystems Web site address is To submit technical questions from North America or Europe: Step Action 1 Access the Applied Biosystems Technical Support Web site. 2 Under the Troubleshooting heading, click Support Request Forms, then select the relevant support region for the product area of interest. 3 Enter the requested information and your question in the displayed form, then click Ask Us RIGHT NOW (blue button with yellow text). 4 Enter the required information in the next form (if you have not already done so), then click Ask Us RIGHT NOW. You will receive an reply to your question from one of our technical experts within 24 to 48 hours. To Obtain Documents on Demand Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available by fax or or by download from our Web site. To order documents... by index number by phone for fax delivery Then... a. Access the Applied Biosystems Technical Support Web site at b. Click the Index link for the document type you want, then find the document you want and record the index number. c. Use the index number when requesting documents following the procedures below. a. From the U.S. or Canada, call , or from outside the U.S. and Canada, call b. Follow the voice instructions to order the documents you want. through the Internet for fax or delivery Note There is a limit of five documents per request. a. Access the Applied Biosystems Technical Support Web site at b. Under Resource Libraries, click the type of document you want. c. Enter or select the requested information in the displayed form, then click Search. d. In the displayed search results, select a check box for the method of delivery for each document that matches your criteria, then click Deliver Selected Documents Now (or click the PDF icon for the document to download it immediately). e. Fill in the information form (if you have not previously done so), then click Deliver Selected Documents Now to submit your order. Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for delivery. Page 15 of 16

16 Copyright 2000, Applied Biosystems For Research Use Only. Not for use in diagnostic procedures. Applied Biosystems, ABI PRISM, and its design are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ABI is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. GeneScan and GeneAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. All other trademarks are the sole property of their respective owners. P/N B, Stock No. 106UB07-02