Sample & Assay Technologies

Transcription

1 July 2008 QIAxcel DNA Handbook QIAxcel DNA High Resolution Kit QIAxcel DNA Screening Kit QIAxcel DNA Large Fragment Kit For automated analysis of DNA fragments using the QIAxcel system Sample & Assay Technologies

2 QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in: Purification of DNA, RNA, and proteins Nucleic acid and protein assays microrna research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit

3 Contents Kit Contents 4 Storage 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 6 Technical Assistance 6 Safety Information 7 Quality Control 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Preparing the QIAxcel gel cartridge and buffer tray 11 Sample preparation recommendations 14 Marker selection 15 Protocol Determining DNA Fragment Sizes and Concentrations Using a QIAxcel DNA Kit with the QIAxcel System 19 Troubleshooting Guide 22 Appendix A: Data Analysis 23 Aligning the gel image 23 Creating a DNA size marker reference table 24 Determination of DNA length and concentration 27 Appendix B: QIAxcel Methods 28 QIAxcel DNA high-resolution methods 28 QIAxcel DNA screening methods 30 QIAxcel DNA large-fragment methods 33 Universal methods: all gel cartridges 35 References 35 Ordering Information 36 QIAxcel DNA Handbook 07/2008 3

4 Kit Contents QIAxcel DNA High Resolution Kit (1200) Catalog no Number of assays 12 x 100 QIAxcel DNA High Resolution Cartridge (with smart key) 1 QX Separation Buffer* QX Wash Buffer* QX Mineral Oil QX DNA Dilution Buffer QX Intensity Calibration Marker 40 ml 40 ml 50 ml 15 ml 600 μl QX 0.2 ml 12-Tube Strips 2 QX Colored 0.2 ml 12-Tube Strips 2 Handbook 1 * Contains sodium azide as a preservative. QIAxcel DNA Screening Kit (2400) Catalog no Number of assays 12 x 200 QIAxcel DNA Screening Cartridge (with smart key) 1 QX Separation Buffer* QX Wash Buffer* QX Mineral Oil QX DNA Dilution Buffer QX Intensity Calibration Marker 40 ml 40 ml 50 ml 15 ml 600 μl QX 0.2 ml 12-Tube Strips 2 QX Colored 0.2 ml 12-Tube Strips 2 Handbook 1 * Contains sodium azide as a preservative. 4 QIAxcel DNA Handbook 07/2008

5 QIAxcel DNA Large Fragment Kit (600) Catalog no Number of assays 12 x 50 QIAxcel DNA Large Fragment Cartridge (with smart key) 1 QX Separation Buffer* QX Wash Buffer* QX Mineral Oil QX DNA Dilution Buffer QX Intensity Calibration Marker 40 ml 40 ml 50 ml 15 ml 600 μl QX 0.2 ml 12-Tube Strips 2 QX Colored 0.2 ml 12-Tube Strips 2 Handbook 1 * Contains sodium azide as a preservative. Storage All components of the QIAxcel DNA Large Fragment Kit, the QIAxcel DNA High Resolution Kit, and the QIAxcel DNA Screening Kit, including gel cartridge, can be stored dry at room temperature (15 25 C). The QIAxcel DNA Large Fragment Kit can be stored in this manner for up to 3 months and the QIAxcel DNA High Resolution Kit and the QIAxcel DNA Screening Kit for up to 6 months. If being used on a daily basis, store the QIAxcel Gel Cartridge in the QIAxcel instrument in the Park position (see the QIAxcel User Manual, Section 5.3). If more than one QIAxcel cartridge were used, store the second cartridge in the cartridge stand filled with wash buffer and covered with oil (see page 5-2 of the QIAxcel User Manual). For long-term storage, return the QIAxcel cartridge to the blister pack, inserting the capillary tips into the soft gel, and store in the dark. Product Use Limitations QIAxcel Kits are intended for research only. Not for use in diagnostic procedures. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN products to adhere to the NIH guidelines QIAxcel DNA Handbook 07/2008 5

6 that have been developed for recombinant DNA experiments, or to other applicable guidelines. Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product as you wish. Separate conditions apply to QIAGEN scientific instruments, service products, and to products shipped on dry ice. Please inquire for more information. A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see back cover or visit Technical Assistance At QIAGEN, we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products. If you have any questions or experience any difficulties regarding the QIAxcel DNA High Resolution Kit, QIAxcel DNA Screening Kit, and QIAxcel DNA Large Fragment Kit or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information, please see our Technical Support Center at or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit 6 QIAxcel DNA Handbook 07/2008

7 Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at where you can find, view, and print the MSDS for each QIAGEN kit and kit component. 24-hour emergency information Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison Information Center Mainz, Germany Tel: Quality Control Each cartridge of the QIAxcel DNA High Resolution Kit, the QIAxcel DNA Screening Kit, and the QIAxcel DNA Large Fragment Kit is tested against predetermined specifications to ensure consistent product quality. QIAxcel DNA Handbook 07/2008 7

8 Introduction The QIAxcel system, when used in conjunction with the QIAxcel DNA High Resolution Kit, the QIAxcel DNA Screening Kit, and the QIAxcel DNA Large Fragment Kit, provides fully automated size separation and quantification of DNA fragments of up to 96 samples per run. QIAxcel technology, based on capillary electrophoresis using gel cartridges, provides unmatched resolution, speed, and throughput. QIAxcel gel cartridges are reusable, allowing up to 200 runs of 12 samples to be performed. Preinstalled methods suitable for most applications are provided. Customized methods can also be created contact QIAGEN Technical Services for more details. The BioCalculator software supplied with the QIAxcel instrument provides both electropherograms and gel images of nucleic acid separation. The QIAxcel RNA Quality Control Kit, which allows RNA separation and quantification on the QIAxcel system, is also available (see Ordering Information on page 36). Principle and procedure The QIAxcel system uses capillary gel electrophoresis to enable fast size-based separation of nucleic acids. Unlike traditional agarose gel electrophoresis, separation is performed in a capillary of a precast gel cartridge. Each sample is automatically loaded (according to voltage and time parameters) into an individual capillary and voltage is applied. The negatively charged nucleic acid molecules migrate through the capillary to the positively charged terminus (Figure 1). As with agarose gel electrophoresis, low-molecular-weight molecules migrate faster than high-molecular-weight molecules. As the molecules migrate though the capillary, they pass a detector that detects and measures the fluorescent signal. A photomultiplier detector converts the emission signal into electronic data, which are then transferred to the computer for further processing using BioCalculator software. After processing, the data are displayed as an electropherogram and a gel image. The QIAxcel system offers a number of advantages over traditional slab-gel electrophoresis, including: Higher detection sensitivity Less sample wastage (minimal sample input volumes) Fast analysis of up to 96 samples Automated loading and analysis 8 QIAxcel DNA Handbook 07/2008

9 Figure 1. Sample separation process using the QIAxcel system. Nucleic acid molecules are size separated by applying an electrical current to a gel-filled capillary. A photomultiplier detector in the QIAxcel instrument detects the nucleic acid molecules as they migrate towards the positively charged terminus of the capillary. The data are converted to an electropherogram and a gel image by the BioCalculator software. QIAxcel DNA Handbook 07/2008 9

10 Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier. Pipets and pipet tips 12-tube strips (e.g., QX 0.2 ml 12-Tube Strip, cat. no ) or 96-well plates Centrifuge with rotor suitable for 0.2 ml strips or 96-well plates, such as the Centrifuge 4-15C or Centrifuge 4K15C* QX Alignment Marker (see Marker selection, page 15) (see Marker selection, page 15) Depending on the sample concentration, it may be necessary to order additional QX DNA Dilution Buffer (cat. no ) * For ordering information, see ). For recommended combinations of QX Alignment Marker and, refer to Table 4, page QIAxcel DNA Handbook 07/2008

11 Important Notes Preparing the QIAxcel gel cartridge and buffer tray Important points before starting The volume of buffer supplied is sufficient for a defined number of runs (see Table 1). If required, additional buffers can be purchased separately (see Ordering Information, page 36). Table 1. Number of runs possible using the supplied buffer volumes Kit Number of runs Samples per run QIAxcel DNA High Resolution Kit QIAxcel DNA Screening Kit QIAxcel DNA Large Fragment Kit The 0.2 ml 12-tube strips containing QX Alignment Marker and QX Intensity Calibration Marker (if required) should fit loosely in the MARKER1 and MARKER2 position (see steps 12 and 16). QX Alignment Markers should be replaced every runs or as needed. Additional markers and buffers may need to be purchased (see Ordering Information, page 36). When not in use, the 12-tube strip containing QX Alignment Marker should be stored at 20 C. Things to do before starting If prepared in advance, the 12-tube strip containing QX Alignment Marker should be equilibrated to operating temperature (20 25ºC) and centrifuged briefly before use. If the QIAxcel gel cartridge is being used for the first time, intensity calibration should be performed (see step 18, page 13). This step is not necessary if the QIAxcel gel cartridge has already been calibrated, unless it is being used on a different QIAxcel instrument or a different computer is used to operate the instrument. If a different computer is used, the calibration log file must be transferred from the computer used to operate the instrument to the new computer so that it is not necessary to run the calibration again. QIAxcel DNA Handbook 07/

12 Procedure Unpacking the QIAxcel gel cartridge 1. Remove all buffer bottles from the kit box. 2. Add 10 ml QX Wash Buffer to both reservoirs of the QX Cartridge Stand (provided with the QIAxcel instrument) and cover with 3 ml mineral oil (supplied). 3. Remove the QIAxcel gel cartridge from its packaging and carefully wipe off any soft gel debris from the capillary tips using a soft tissue. 4. Remove the purge cap seal from the back of the QIAxcel gel cartridge and place it in the QX Cartridge Stand. Note: A soft tissue should be used to wipe off any gel that may have leaked from the purge cap port. Note: Ensure that the capillary tips are submerged in QX Wash Buffer. 5. Incubate new cartridges in the QX Cartridge Stand for 20 min prior to use. Preparing the buffer tray 6. Allow all reagents to equilibrate to room temperature (15 25 C) before use. 7. Wash the buffer tray with hot water and rinse thoroughly with deionized water. 8. Fill the WP and WI positions of the buffer tray with 8 ml QX Wash Buffer. 9. Fill the BUF position of the buffer tray with 18 ml QX Separation Buffer. 10. Carefully add mineral oil all 3 positions to prevent evaporation: add 2 ml mineral oil to positions WP and WI and 4 ml mineral oil to position BUF. All 3 positions should be covered with mineral oil. 11. Insert the buffer tray into the buffer tray holder so that the slots for the 12-tube strips face the front of the instrument. Preparing QX Alignment Markers 12. Load 15 μl QX Alignment Marker into each well of a QX 0.2 ml 12-Tube Strip. 13. Add 1 drop of mineral oil to each well, and insert the strip into the MARKER1 position of the buffer tray. Important: The 12-tube strip should fit loosely in the MARKER1 position on the buffer tray. 12 QIAxcel DNA Handbook 07/2008

13 Installing a QIAxcel gel cartridge and smart key 14. Remove the QIAxcel gel cartridge from the QX Cartridge Stand. 15. Open the cartridge door and insert the QIAxcel gel cartridge into the QIAxcel system. The cartridge description label should face the front and the purge hole should face the back of the system. 16. Insert the smart key into the smart key socket. The smart key can be inserted in either direction. 17. Close the cartridge door. The cartridge ID, number of runs remaining, and cartridge type will be displayed automatically in the Instrument Control window once the smart key is latched. Note: The system will not recognize the cartridge and will not operate if the smart key is not inserted. Intensity calibration Every QIAxcel cartridge requires intensity calibration prior to sample analysis. The intensities of each capillary are normalized and a factor is applied for every subsequent run. This corrects for natural intensity reading variations between each capillary in the cartridge. The data (individual calibration data files) for each cartridge intensity calibration are stored in the CALdata folder. This folder is saved in the BioCalculator root diretory: C:\Program Files\QIAxcel BioCalculator. A Calibration2.log file (cartridge calibration information) will be saved automatically in the BioCalculator root directory: C:\Program Files\QIAxcel BioCalculator. If for any reason a different computer is used than the one storing the calibration2.log file, the calibration2.log file should be transferred to the new computer. Otherwise, recalibration of the cartridge is required. Likewise, if the QIAxcel gel cartridge is used on a different QIAxcel instrument than the one it was calibrated on, another intensity calibration should be performed. Running the calibration wizard Important: The 12-tube strip should fit snugly in the MARKER2 position on the buffer tray. This process takes about 16 min. 18. Load 15 μl of Intensity Calibration Marker solution into each well of a QX Colored 0.2ml 12-Tube Strip, make sure no air bubbles are trapped in the solution, and insert it into the Marker 2 position of the buffer tray. QIAxcel DNA Handbook 07/

14 19. Launch the calibration wizard by selecting File then Intensity Calibration in the Instrument Control window. 20. Click Start to begin the cartridge intensity calibration. 21. Once the calibration is complete, The Calibration Verification dialog box will open. This will show either a Pass or Fail for each channel. Note: A successful calibrated cartridge should have normalized area calibrated range between If one or more channels fail, the calibration process should be repeated and new strip of Intensity Calibration Marker solution must be used. 23. If one or more channels showed no signals in the first run, call QIAGEN Technical Service. 24. If one or more channels showed high background, see Section 8 in the QIAxcel User Manual. 25. If calibration failed more than once, call QIAGEN Technical Service. Recalibration process Note: A new strip of Intensity Calibration Marker solution should be used for each recalibration run. Re-using the intensity calibration marker may have an impact on the calibration data and can cause them to be out of range. 26. Load a new strip of QX Intensity Calibration Marker, make sure there are no air bubbles, and insert the strip onto the buffer tray marker 2 position. 27. Launch the calibration wizard by selecting File then Intensity Calibration in the Instrument Control window. 28. Click Recalibrate, and then Start to repeat the calibration routine. DNA size marker Once created, a single DNA reference marker table can be used for the entire life of the cartridge (see Appendix A, page 23). However, variations in separation temperature of the cartridge and buffer can introduce variations in DNA size determination. For optimal results, we recommend creating a new DNA reference marker table every 8 runs or after each 96-well plate. Sample preparation recommendations The minimum sample volume required for analysis is 10 μl. Less than 0.1 μl of the sample will be loaded onto the QIAxcel gel cartridge for analysis. The remaining DNA can be kept for reanalysis or downstream processing. 14 QIAxcel DNA Handbook 07/2008

15 Sample concentration If less than 12 samples are to be processed, the empty wells should be filled with QX DNA Dilution Buffer or a buffer that has a salt concentration similar to the samples. Failure to do this may cause damage to the capillaries. Unpurified DNA samples, such as PCR products (including multiplex PCR), STR, microsatellites, or purified circular plasmid DNA can be directly analyzed without prior purification on the QIAxcel system using the M methods (see Appendix B, page 28). It is not necessary to dilute PCR products prior to analysis. However, other DNA samples with a concentration of >100 ng/μl should be diluted 1:2 (e.g., by adding 5 μl DNA to 5 μl QX DNA Dilution Buffer) or higher to obtain unsaturated signals. Alternatively, the H methods can be used (see Appendix B, page 28). Purified plasmid samples with a concentration of >300 ng/μl should be diluted 1:10 in QX DNA Dilution Buffer (e.g., by adding 1 μl DNA to 9 μl QX DNA Dilution Buffer). In addition, the APL600 method should be used with an adjusted sample injection time to obtain optimal signals. Salt content DNA samples containing a high salt concentration (e.g., restriction digested DNA and plasmid DNA with >10 mm NaCl) may need to be diluted 1:2 with QX DNA Dilution Buffer (e.g., by adding 5 μl DNA to 5 μl QX DNA Dilution Buffer). High salt concentration can inhibit DNA injection, leading to reduced peak heights. High resolution analysis For high-resolution DNA analysis (i.e., 3 5 bp), we recommend adjusting the sample injection time to obtain a signal height of approximately 0.5 RFU (Relative Fluorescence Units). Marker selection Alignment marker For optimal DNA fragment size determination, select a QX Alignment Marker with a fragment size close to the size of your samples. A product finder is available at to help with selection. For example, if analyzing a sample consisting of DNA fragments that are bp in size, the QX Alignment Marker 15bp/500bp (cat. no ) should be used. For general applications, the QX Alignment Marker 15 bp/5 kb (cat. no ) is suitable when using the QIAxcel DNA High Resolution Kit or the QIAxcel DNA Screening Kit. If using the QIAxcel DNA Large Fragment Kit, the QX Alignment QIAxcel DNA Handbook 07/

16 Marker 15 bp/10 kb is suitable for general use. Table 2 provides a complete overview of available QX Alignment Markers. Table 2. QX Alignment Markers Product Cat. no. QX Alignment Marker 15 bp/500 bp (1.5 ml) QX Alignment Marker 15 bp/1 kb (1.5 ml) QX Alignment Marker 15 bp/3 kb (1.5 ml) QX Alignment Marker 15 bp/10 kb (1.5 ml) QX Alignment Marker 15 bp/5 kb (1.5 ml) QX Alignment Marker 50 bp/500 bp (1.5 ml) QX Alignment Marker 50 bp/1 kb (1.5 ml) QX Alignment Marker 15 bp/400 bp (1.5 ml) QX Alignment Marker 50 bp/3 kb (1.5 ml) QX Alignment Marker 50 bp/5 kb (1.5 ml) DNA size marker BioCalculator software calculates both the DNA fragment size and concentration based on the fragment migration time and the area of each peak in comparison to a reference. The DNA size (bp) is calculated using a point-to-point calculation using 2 DNA size marker fragments. To enable accurate size and concentration measurements, the DNA fragments to be analyzed must fall within the smallest and largest fragment sizes of the chosen. For optimal DNA size determination, select a with fragments closest to the size of your DNA sample. For optimal concentration determination, we recommend diluting the in the same buffer that was used for the DNA samples. For undiluted DNA samples, we recommend diluting the 100 ng/μl in 1x PCR buffer or 1x restriction digestion buffer to the following final concentration: 10 ng/μl for L methods 30 ng/μl for M methods 50 ng/μl for H methods For samples diluted in QX DNA Dilution Buffer, we recommend diluting the DNA size marker to a final concentration of 5 ng/μl in QX DNA Dilution Buffer. 16 QIAxcel DNA Handbook 07/2008

17 Table 3 provides a complete overview of the available s. Table 4 provides suggestions for combining s and QX Alignment Markers. Table 3. s Product Fragment sizes (bp) Cat. no. puc18/haeiii (50 μl) 80, 102, 174, 257, 267, 298, 434, 458, FX174/HaeIII (50 μl) 25 bp 1.8 kb (50 μl) 100 bp 3 kb (50 μl) bp (50 μl) bp (50 μl) 250 bp 4 kb (50 μl) 250 bp 8 kb (50 μl) 72, 118, 194, 234, 271, 281, 310, 603, 872, 1078, , 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 1.8 kb* 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, , 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 1.8 kb* 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, , 500, 750, 1000, 1200, 1500, 2000, 2500, 3000, 3500, , 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, * The 1.8 kb fragment has 10 times the intensity of other bands and, as such, cannot be used for all methods. Only methods OM700, OL700, or OH700 are suitable for use with these markers as the 1.8 kb band will not appear in the run. QIAxcel DNA Handbook 07/

18 Table 4. Suggested and QX Alignment Marker combinations Size marker Cat. no. Alignment marker Cat. no. puc18/haeiii (50 μl) QX Alignment Marker 15 bp/1 kb (1.5 ml) FX174/HaeIII (50 μl) 25 bp 1.8 kb (50 μl) 100 bp 3 kb (50 μl) bp (50 μl) bp (50 μl) 250 bp 4 kb (50 μl) 250 bp 8 kb (50 μl) QX Alignment Marker 15 bp/3 kb (1.5 ml) QX Alignment Marker 15 bp/400 bp (1.5 ml) QX Alignment Marker 15 bp/5 kb (1.5 ml) QX Alignment Marker 15 bp/500 bp (1.5 ml) QX Alignment Marker 15 bp/1 kb (1.5 ml) QX Alignment Marker 50 bp/5 kb (1.5 ml) QX Alignment Marker 15 bp/10 kb (1.5 ml) Method selection A number of preinstalled methods are available for each QIAxcel gel cartridge. To select the appropriate method for the samples being analyzed, refer to Appendix B, page QIAxcel DNA Handbook 07/2008

19 Protocol: Determining DNA Fragment Sizes and Concentrations Using a QIAxcel DNA Kit with the QIAxcel System Important points before starting Before beginning the procedure, read Important Notes starting on page 11. Make sure to select QX Alignment and DNA Size Markers with the appropriate fragment sizes (see Marker selection starting on page 15 or visit QXmarker). For optimal results, the solution containing the DNA samples should be approximately ph 6 9 and should not have an ionic content greater than that of a typical PCR buffer. Determine the optimal QIAxcel method for sample analysis (see Appendix B, page 28). Things to do before starting Ensure samples have been prepared according to the instructions in Sample preparation recommendations, page 14. Ensure that the QIAxcel gel cartridge is set up and all reagents have been prepared according to the instructions in Preparing the QIAxcel gel cartridge and buffer tray, page 11. Optional: Create a DNA reference marker table before running samples (see Appendix A on page 23 for more details). The table can be prepared after the sample run, if required. Procedure 1. Switch on the QIAxcel system at the power switch. 2. Switch on the computer and launch the BioCalculator software. 3. Install the QIAxcel gel cartridge. See the QIAxcel User Manual for more details. QIAxcel DNA Handbook 07/

20 4. Load the buffer tray containing the QX Alignment Marker into the buffer tray holder. See the QIAxcel User Manual for more details. Note: If being used for the first time, the QIAxcel gel cartridge will require calibration (see Preparing the QIAxcel gel cartridge and buffer tray page 13). Note: QX Alignment Markers should be replaced every runs or as needed. QX Alignment Marker should be stored at 20 C between uses. 5. Load the sample strips in position A or load the 96-well plate containing samples onto the sample tray holder. Note: The cartridge door and sample door of the QIAxcel system must remain closed during operation of the instrument. Opening the cartridge door or sample door during operation will cause the system to stop any action it is currently performing. 6. Select the appropriate method in the Instrument Control window. Refer to see Appendix B, page 28 for more details on the preinstalled methods. 20 QIAxcel DNA Handbook 07/2008

21 7. Enter the sample name, position, and number of runs in the corresponding fields of the Instrument Control window. 8. Optional: Enter the sample injection time (minimum: 1 s; maximum: 60 s) in the time column. When left blank, the default settings for the method chosen are used. 9. To perform multiple analyses of the same row, enter the number of repeats in the Runs field. To run a 96-well plate, check the increments box ( Inc ) and enter 8 in the Runs field. Note: The same method and injection time will apply to all runs. 10. Select the data directory where the run should be stored. Note: Subfolders can be created in the data directory by optionally entering the user and plate identifiers in the corresponding dialog box fields. Optional: If connected to a network (in addition to a local data directory), a network directory may be chosen for saving the run data. 11. Recommended: Click the Sample Info button to enter sample information for each well. Alternatively, sample information in a spreadsheet can be imported in *.csv file format. 12. Make sure that the separation channels to be used are checked (i.e., if running less than the maximum number samples, check only the channels that contain samples). Note: Unused wells should contain QX DNA Dilution Buffer to prevent damage to the channel. 13. Optional: Check Automatically analyze after data acquisition. When this option is checked, the data will be analyzed according to the parameter settings automatically after the raw data has been acquired. 14. Optional: Check Include reference marker table. When this option is checked, DNA size determination is performed automatically. See Appendix A, page 23, for more information. 15. Click the Marker button and open the desired DNA reference marker. Ensure that the same method used to create the DNA reference marker table is used for the sample analysis being performed (see Appendix A, page 23). QIAxcel DNA Handbook 07/

22 Troubleshooting Guide Section 8 of the QIAxcel User Manual contains a troubleshooting guide, which may be helpful in solving any problems that might arise. In addition, extensive user information is also provided in the Help menu of the BioCalculator software. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies (for contact information, see back cover or visit 22 QIAxcel DNA Handbook 07/2008

23 Appendix A: Data Analysis Aligning the gel image After the run, a gel image window is displayed. This is referred to as the Folder View (Figure 2). An additional window within the Folder View window displays data for all channels. Data from an individual channel can be opened by double-clicking. All of the data generated are automatically aligned by the BioCalculator software; however, manual alignment is also possible (see procedure below). Alignment compensates for any slight variations between the different capillaries and results in the alignment of the first and last peak (band) across all 12 channels. Figure 2. The "Folder View". QIAxcel DNA Handbook 07/

24 Manual alignment procedure 1. Open the Parameter setup dialog box by clicking Analysis and then Parameters in the BioCalculator menu bar. 2. In the Data Smoothing filter (pts) drop-down menu, select In the Markers section, check First peak and Last peak. 4. Check Use as default, and click OK. 5. Select Analysis and then Run in the BioCalculator menu bar. If the peaks or bands are not aligned, refer to Section 8 in the QIAxcel User Manual for further help. Creating a DNA size marker reference table The DNA size marker reference table allows accurate size determination of sample DNA fragment sizes. A single DNA size marker reference table can be used for the entire life of the cartridge. However, the separation temperature and age of cartridge and buffer will introduce variations into DNA size determination. For optimal results, we recommend creating a new DNA reference marker table every 8 runs or after each 96-well plate. The DNA size marker reference table allows the size of sample DNA fragments to be determined without the need to run a every time. 24 QIAxcel DNA Handbook 07/2008

25 Important points before starting Only one channel or capillary is required to generate the DNA size marker reference table. The relative migration time (reltime) of the DNA size marker reference table is dependant on the separation voltage. Thus, the same separation voltage used to create the DNA size marker reference table must be used for the sample run (i.e., the 0M500 DNA size marker reference table can be used for 0M500, 0H500, or 0L500 methods as these methods use the same separation voltage). The normalized area of the peak is dependant on the injection voltage and the injection time. Thus, the same injection conditions used to create the DNA size marker reference table must be used for the sample run (i.e., the 0M500 DNA size marker reference table can be used for 0M400 methods). For STR and microsatellite genotyping, a DNA size marker should be included in each sample run. The DNA size marker should be diluted in the same buffer as the sample to obtain optimal results (see Sample preparation recommendations page 14). DNA size marker reference table procedure 1. Open the data acquired for the DNA size marker channel by selecting the corresponding file from the file bar or by clicking the title bar of the corresponding dialog box and maximize the window. In the results table at the bottom of the dialog channel 1 box, ensure all peaks are above the positive threshold setting. 2. Select Analysis and then Reference Markers from the BioCalculator menu bar to open the Reference Markers dialog box. 3. Click Size/Conc. From the drop-down menu. 4. Click Open to select a reference marker file. A list of reference s is shown. Note: A custom DNA size marker reference table (i.e., allowing use of different DNA size markers) can be created by clicking New and entering the relevant information. 5. Double-click the name of the reference DNA size marker file to be opened. Alternatively, select the file to be opened, and click the Open button. The selected reference data are shown in the Reference Marker dialog box. QIAxcel DNA Handbook 07/

26 6. Position the cursor in the first row of the table and click Insert to enter a new (blank) row above the existing rows. 7. Enter the first peak value of the QX Alignment Marker (i.e., 15 bp for the marker with cat. no ) in the Size (bp) field. 8. Position the cursor in the last row of the table and click Insert to enter a new (blank) row below the existing rows. 9. Enter the last peak value of the QX Alignment Marker (i.e., 5000 bp for the marker with cat. no ) in the Size (bp) field. 10. Click both Copy buttons (above Reltime and NA ) to copy the relative migration time and normalized area from the result table to the reference marker table. 11. Check Apply to all documents. Note: Always ensure that the value for the first peak of the alignment marker in the Reltime column is 0 and the value for the last peak of the alignment marker is 1. The number of peaks in the data table acquired for channel 1 must be the same as the number of peaks in the reference marker table. Otherwise, the Reltime column will show an incorrect number of rows in the reference marker table, which will result in miscalculation of the DNA size and concentration. 12. Click Save. To use the reference marker for future analysis, we recommend saving the file under a different file name that includes the name of the method used. 26 QIAxcel DNA Handbook 07/2008

27 13. Select Analysis/Run from the menu bar. The fragment size and concentration will appear for each DNA fragment in each channel. Determination of DNA length and concentration Gel image format The fragment sizes (bp) of the reference marker are displayed on both sides of the gel image window. In addition, the estimated sizes of the sample DNA fragments are shown. DNA fragment sizes are displayed when the mouse cursor is moved over a fragment. Individual data The size (bp) of the DNA fragment appears next to each peak in the electropherogram and in the Size (bp) column of the results table. The concentration of the DNA fragment appears only in the Conc (ng/μl) column of the results table. A size and concentration of 0 is shown for the first and last peak of the QX Alignment Marker. QIAxcel DNA Handbook 07/

28 Appendix B: QIAxcel Methods QIAxcel DNA high-resolution methods The QIAxcel DNA High Resolution Cartridge is designed for high-resolution (3 5 bp) genotyping, high-resolution multiplex PCR, and AFLP/RFLP analysis of less than 20 fragments. The gel cartridge can separate fragment sizes ranging from 15 bp to 5 kb. The resolution depends on the fragment size and the method chosen to run the assay (see Tables 5 to 8). Table 5. Guidelines for method selection using the QIAxcel DNA High Resolution Kit Method 0M400* 0L400 0H400 0M500* 0L500 0H500 0M700* 0L700 0H700 Fragment size bp 500 bp 1 kb 1 5 kb Best resolution 20 bp 100 bp 500 bp 10 bp 50 bp 200 bp 3 5 bp N/A N/A * The 0M400, 0M500, and 0M700 methods are recommended for DNA concentrations of ng/μl (e.g., PCR products amplified from genomic DNA with cycles). The 0L400, 0L500, and 0L700 methods are recommended for DNA concentrations of <10 ng/μl. The 0H400, 0H500, and 0H700 methods are recommended for DNA concentrations of >100 ng/μl (e.g., high-yield PCR products). 28 QIAxcel DNA Handbook 07/2008

29 M methods The 0M400, 0M500, and 0M700 methods are recommended for DNA concentrations of ng/μl. Table 6. M methods for the QIAxcel DNA High Resolution Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) 0M M M * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. L methods The 0L400, 0L500, and 0L700 methods are recommended for DNA concentrations of <10 ng/μl. Table 7. L methods for the QIAxcel DNA High Resolution Kit Sample injection Method voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) 0L L L * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. QIAxcel DNA Handbook 07/

30 H Methods The 0H400, 0H500, and 0H700 methods are recommended for DNA concentrations of >100ng/μl. Table 8. H methods for the QIAxcel DNA High Resolution Kit Sample injection Method voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) 0H H H * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. Note: Unpurified PCR products contain dntps and primers, which can contribute to the optical density (OD) and cause over estimation of the DNA concentration. QIAxcel DNA screening methods The QIAxcel DNA Screening Kit is designed for low-resolution (>20bp) genotyping, low-resolution multiplex PCR, PCR screening, analysis of plasmid DNA restriction digests, and determination of amount of plasmid or oligo DNA. The gel cartridge can separate fragments from 15 bp to 5 kb. The resolution depends on the fragment size and the method chosen to run the assay (see Tables 9 to 12). 30 QIAxcel DNA Handbook 07/2008

31 Table 9. Guidelines for method selection using the QIAxcel DNA Screening Kit Method AM320* AL320 AH320 AM420* AL420 AH420 APH600 APL600 Fragment size <500 bp 500 bp 1 kb 1 5 kb Best resolution 20 bp 100 bp 500 bp 20 bp 100 bp 500 bp Analysis of uncut plasmid DNA * The AM320 and AM420 methods are recommended for DNA concentrations of ng/μl (e.g., PCR products amplified from genomic DNA with cycles). The AL320 and AL420 methods are recommended for DNA concentrations of <10 ng/μl. The AH320 and AH420 methods are recommended for DNA concentrations of >100 ng/μl (e.g., high-yield PCR products). M methods The AM320 and AM420 methods are recommended for DNA concentrations of ng/μl, and method APH600 is recommended for purified high-copy plasmid DNA ( ng/μl) in elution buffer. Table 10. M methods for the QIAxcel DNA Screening Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) AM AM APH * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. QIAxcel DNA Handbook 07/

32 L Methods The AL320 and AL420 methods are recommended for DNA concentrations of <10 ng/μl, and method APL600 is recommended for purified low-copy plasmid DNA (<50 ng/μl) in elution buffer. Table 11. L methods for the QIAxcel DNA Screening Kit Sample Method injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) AL AL APL * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. H Methods The AL320 and AL420 methods are recommended for DNA concentrations of >100ng/μl. Table 12. H methods for the QIAxcel DNA Screening Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) AH AH * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. Note: Unpurified PCR products contain dntps and primers, which can contribute to the optical density (OD) and cause overestimation of the DNA concentration. 32 QIAxcel DNA Handbook 07/2008

33 QIAxcel DNA large-fragment methods The QIAxcel DNA Large Fragment Kit is designed for separating small and large DNA fragments between 5 kb and10 kb. The resolution depends on the fragment size and the method chosen to run the assay (see Tables 13 to 16). Table 13. Guidelines for method selection using the QIAxcel DNA Large Fragment Kit Method BM800* BL800 BH800 BM1200* BL1200 BH1200 Fragment size bp 500 bp 1 kb 1 5 kb 5 10 kb Best resolution 3 5 bp 50 bp 100 bp N/A 3 5 bp 50 bp 100 bp 500 bp * The BM800 and BM1200 methods are recommended for DNA concentrations of ng/μl (e.g., PCR products [30 40 cycles] amplified from genomic DNA). The BL800 and BL1200 methods are recommended for DNA concentrations of <10 ng/μl. The BH800 and BH1200 methods are recommended for DNA concentrations of >100 ng/μl (e.g., high-yield PCR products). M methods The BM800 and BM1200 methods are recommended for DNA concentrations of ng/μl. Table 14. M methods for the QIAxcel DNA Large Fragment Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) BM BM * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. QIAxcel DNA Handbook 07/

34 L methods The BL1200 and BL800 methods are recommended for DNA concentrations of <10 ng/μl. Table 15. L methods for the QIAxcel DNA Large Fragment Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) BL BL * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. H methods The BH1200 and BH800 methods are recommended for DNA concentrations of >100ng/μl. Table 16. H methods for the QIAxcel DNA Large Fragment Kit Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) BH BH * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. Note: Unpurified PCR products contain dntps and primers, which can contribute to the optical density (OD) and cause over estimation of the DNA concentration. 34 QIAxcel DNA Handbook 07/2008

35 Universal methods: all gel cartridges There are 2 built-in universal methods that can be used with all QIAxcel gel cartridges (Table 17). The Purge method allows the user to remove air bubbles from the gel cartridge and clean the capillaries. The Purge method is not counted as a sample run, so the number of sample runs remaining is unchanged. The HVpurge method applies a high voltage (8 kilovolts) and N 2 pressure to the capillaries to correct misalignment or delays in sample migration through the capillaries. The HVpurge method is not counted as a sample run so the number of sample runs remaining is unchanged. Note: Do not exceed a total of 10 runs (or 5 minutes) when using either a Purge or HVpurge method for any cartridge, with the exception of the QIAxcel DNA Screening Cartridge, which should not exceed a total of 5 runs or 2 3 minutes. In addition, to prevent excessive heat build-up within the gel cartridge, do not exceed more than 3 consecutive uses of the HVpurge method. Table 17. Methods for all gel cartridges Method Sample injection voltage (KV) Sample injection time (s)* Separation voltage (KV) Separation time (s) Purge N/A N/A N/A 30 HVpurge N/A N/A 8 30 * Sample injection time can be adjusted from 5 to 40 seconds to obtain optimal signals. Reduce the injection time if the signal is saturated, as indicated by peaks with flat tops. Increase sample injection time if the signal is below the default setting of the positive threshold of 7%. N/A: not applicable References QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc. For a complete list of references, visit the QIAGEN Reference Database online at or contact QIAGEN Technical Services or your local distributor. QIAxcel DNA Handbook 07/

36 Ordering Information Product Contents Cat. no. QIAxcel System Capillary electrophoresis device, including computer, and BioCalculator Analysis software; 1-year warranty on parts and labor Warranty PLUS 2, QIAxcel QIAxcel Kits QIAxcel DNA High Resolution Kit (1200) QIAxcel DNA Screening Kit (2400) QIAxcel DNA Large Fragment Kit (600) QIAxcel RNA Quality Control Kit (1200) Software BioCalculator Software DNA size markers puc18/haeiii (50 μl) FX174/HaeIII (50 μl) 25 bp 1.8 kb (50 μl) 3-year warranty, 5-working day response time, all labor, travel, and repair parts QIAxcel DNA High Resolution Gel Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, 12-Tube Strips QIAxcel DNA Screening Gel Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, 12-Tube Strips QIAxcel DNA Large Fragment Gel Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, 12-Tube Strips QIAxcel RNA Quality Control Gel Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, QX Alignment Marker, 12-Tube Strips Separate license for use of BioCalculator analysis software on an additional computer DNA size marker with 9 fragments: bp DNA size marker with 11 fragments: bp DNA size marker with 12 fragments: 25 bp 1.8 kb QIAxcel DNA Handbook 07/2008

37 Product Contents Cat. no. 100 bp 3 kb (50 μl) DNA size marker with 14 fragments: 100 bp 3 kb bp (50 μl) bp (50 μl) 250 bp 4 kb (50 μl) 250 bp 8 kb (50 μl) Alignment markers QX Alignment Marker 15 bp/500 bp (1.5 ml) QX Alignment Marker 15 bp/1 kb (1.5 ml) QX Alignment Marker 15 bp/3 kb (1.5 ml) QX Alignment Marker 15 bp/10 kb (1.5 ml) QX Alignment Marker 15 bp/5 kb (1.5 ml) QX Alignment Marker 50 bp/500 bp (1.5 ml) QX Alignment Marker 50 bp/1 kb (1.5 ml) QX Alignment Marker 15bp/400bp (1.5 ml) QX Alignment Marker 50 bp/3 kb (1.5 ml) QX Alignment Marker 50 bp/5 kb (1.5 ml) DNA size marker with 17 fragments: bp DNA size marker with 11 fragments: bp DNA size marker with 11 fragments: 250 bp 4 kb DNA size marker with 11 fragments: 250 bp 8 kb Alignment marker with 15 bp and 500 bp fragments Alignment marker with 15 bp and 1 kb fragments Alignment marker with 15 bp and 3 kb fragments Alignment marker with 15 bp and 10 kb fragments Alignment marker with 15 bp and 5 kb fragments Alignment marker with 50 bp and 500 bp fragments Alignment marker with 50 bp and 1 kb fragments Alignment marker with 15 bp and 400 bp fragments Alignment marker with 50 bp and 3 kb fragments Alignment marker with 50 bp and 5 kb fragments QIAxcel DNA Handbook 07/

38 Product Contents Cat. no. QX RNA Alignment Marker (1.5 ml) Calibration marker RNA alignment marker QX Intensity Calibration Marker (600 μl) 600 μl QX Intensity Calibration Marker Buffers QX DNA Dilution Buffer (15 ml) QX RNA Dilution Buffer (15 ml) QX Separation Buffer (40 ml) QX Wash Buffer (40 ml) QX Mineral Oil (50 ml) 15 ml QX DNA Dilution Buffer ml QX RNA Dilution Buffer ml QX Separation Buffer ml QX Wash Buffer ml QX Mineral Oil Accessories QX Cartridge Stand QX Cartridge Stand QX Buffer Tray QX Buffer Tray QX 0.2 ml 12-Tube Strip (80) QX Color 0.2 ml 12-Tube Strip (80) QX Nitrogen Cylinder (6) 80 x QX 0.2 ml 12-Tube Strips x QX Color 0.2 ml 12-Tube Strips x QX Nitrogen Cylinder QIAxcel DNA Handbook 07/2008

39 Trademarks: QIAGEN (QIAGEN Group). Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the QIAxcel DNA High Resolution Kit, the QIAxcel DNA Screening Kit, or the QIAxcel DNA Large Fragment Kit to the following terms: 1. The QIAxcel DNA High Resolution Kit, the QIAxcel DNA Screening Kit, or the QIAxcel DNA Large Fragment Kit may be used solely in accordance with the QIAxcel DNA Handbook and for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the QIAxcel DNA Handbook and additional protocols available at 2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties. 3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold. 4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. 5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components. For updated license terms, see QIAGEN, all rights reserved.

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