Agilent DNA 7500 and DNA 12000 Kit Guide Agilent Technologies
Notices © Agilent Technologies, Inc. 2000, 2000-2006, 2013 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. Manual Part Number G2938-90024 Rev.
Contents 1 Agilent DNA 7500 and DNA 12000 Kit 4 2 Equipment Required for a DNA 7500 and DNA 12000 Assay 3 Setting up the Assay Equipment and Bioanalyzer Setting up the Chip Priming Station Setting up the Bioanalyzer Vortex Mixer 6 7 8 8 Starting the 2100 Expert Software 9 4 Essential Measurement Practices 5 Agilent DNA 7500 and DNA 12000 Assay Protocol Preparing the Gel-Dye Mix Loading the Gel-Dye Mix Loading the Marker 10 13 14 15 Inserting a Chip in the Agilent 2100 Bioanalyzer Startin
Agilent DNA 7500 and Agilent DNA 7500 and DNA 12000 Kit Agilent DNA 7500 Kit (reorder number 5067-1506) DNA Chips 25 DNA Chips 1 Electrode Cleaner DNA 7500 Reagents (reorder number 5067-1507)& Supplies (yellow) DNA 7500 Ladder (green) DNA 7500 Markers (2 vials) (blue) DNA Dye Concentrate*(1 vial) (red) DNA Gel Matrix (3 vials) 3 Spin Filters Syringe Kit 1 Syringe Agilent DNA 12000 Kit (reorder number 5067-1508) DNA Chips 25 DNA Chips 1 Electrode Cleaner DNA 12000 Reagents (reorder number 6067-150
Agilent DNA 7500 and Equipment Required for a DNA 7500 and DNA 12000 Assay Equipment Supplied with the Agilent 2100 Bioanalyzer • Chip priming station (reorder number 5065- 4401) • IKA vortex mixer Additional Material Required (Not Supplied) • Pipettes (10 µl, 100 µl and 1000 µl) with compatible tips • Microcentrifuge tubes: • 0.5 ml for sample preparation • 1.5 ml for gel- dye mix preparation • Microcentrifuge Check the Agilent Lab- on- a- Chip webpage for details on assays: www.agilent.
Agilent DNA 7500 and Setting up the Assay Equipment and Bioanalyzer Before beginning the chip preparation protocol, ensure that the chip priming station and the bioanalyzer are set up and ready to use.
Setting up the Assay Equipment and Bioanalyzer Setting up the Chip Priming Station 3 Setting up the Chip Priming Station NOTE Replace the syringe with each new reagent kit. 1 Replace the syringe: a Unscrew the old syringe from the lid of the chip priming station. b Release the old syringe from the clip. Discard the old syringe. c Remove the plastic cap of the new syringe and insert it into the clip. d Slide it into the hole of the luer lock adapter and screw it tightly to the chip priming station.
3 Setting up the Assay Equipment and Bioanalyzer Setting up the Bioanalyzer Setting up the Bioanalyzer Adjust the chip selector: 1 Open the lid of the bioanalyzer and make sure that the electrode cartridge is inserted in the instrument. If not, open the latch, remove the pressure cartridge and insert the electrode cartridge. 2 Remove any remaining chip and adjust the chip selector to position (1). Vortex Mixer IKA - Model MS3 To set up the vortex mixer, adjust the speed knob to 2400 rpm.
Setting up the Assay Equipment and Bioanalyzer Starting the 2100 Expert Software 3 Starting the 2100 Expert Software To start the software: 1 Go to your desktop and double- click the following icon. The screen of the software appears in the Instrument context.
Agilent DNA 7500 and Essential Measurement Practices • Handle and store all reagents according to the instructions on the label of the individual box. • Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results. • Keep all reagent and reagent mixes refrigerated at 4 °C when not in use. • Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use.
Agilent DNA 7500 and Agilent DNA 7500 and DNA 12000 Assay Protocol After completing the initial steps in “Setting up the Assay Equipment and Bioanalyzer” on page 6, you can prepare the assay, load the chip, and run the assay, as described in the following procedures. Preparing the Gel-Dye Mix WA R N I N G Handling DMSO Kit components contain DMSO. Because the dye binds to nucleic acids, it should be treated as a potential mutagen and used with appropriate care.
5 Agilent DNA 7500 and DNA 12000 Assay Protocol Preparing the Gel-Dye Mix 2 Vortex the blue- capped vial with DNA dye concentrate (blue ) for 10 seconds and spin down. Make sure the DMSO is completely thawed. gel-dye mix 25 µl dye 3 Pipette 25 µl of the dye concentrate (blue ) into a DNA gel matrix vial (red ). Store the dye concentrate at 4 °C in the dark again. NOTE Always use the volumes indicated. Using different volumes in the same ratio will produce inaccurate results.
Agilent DNA 7500 and DNA 12000 Assay Protocol Loading the Gel-Dye Mix 5 Loading the Gel-Dye Mix NOTE Before loading the gel-dye mix, make sure that the base plate of the chip priming station is in position (C) and the adjustable clip is set to the highest position. Refer to “Setting up the Chip Priming Station” on page 7 for details. 1 Allow the gel- dye mix to equilibrate to room temperature for 30 minutes before use. Protect the gel- dye mix from light during this time.
5 Agilent DNA 7500 and DNA 12000 Assay Protocol Loading the Marker 5 Press the plunger of the syringe down until it is held by the clip. 6 Wait for exactly 30 seconds and then release the plunger with the clip release mechanism. 7 Visually inspect that the plunger moves back at least to the 0.3 ml mark. pressurize 8 Wait for 5 seconds, then slowly pull back the plunger to the 1 ml position. 9 Open the chip priming station. 10 Pipette 9.
Agilent DNA 7500 and DNA 12000 Assay Protocol Loading the Ladder and the Samples 5 Loading the Ladder and the Samples 1 Pipette 1 µl of the yellow- capped DNA ladder vial (yellow ) in the well marked with the ladder symbol . 1 µl ladder 2 In each of the 12 sample wells pipette 1 µl of sample (used wells) or 1 µl of deionized water (unused wells).
5 Agilent DNA 7500 and DNA 12000 Assay Protocol Inserting a Chip in the Agilent 2100 Bioanalyzer Inserting a Chip in the Agilent 2100 Bioanalyzer 1 Open the lid of the Agilent 2100 bioanalyzer. 2 Check that the electrode cartridge is inserted properly and the chip selector is in position (1). Refer to “Setting up the Bioanalyzer” on page 8 for details. 3 Place the chip carefully into the receptacle. The chip fits only one way. 4 Carefully close the lid.
Agilent DNA 7500 and DNA 12000 Assay Protocol Starting the Chip Run 5 Starting the Chip Run NOTE Please note that the order of executing the chip run may change if the Agilent Security Pack software (only applicable for Agilent 2100 expert software Revision B.02.02 and higher) is installed. For more details please read the 'User's Guide' which is part of the Online Help of your 2100 expert software. 1 In the Instrument context, select the appropriate assay from the Assay menu.
5 Agilent DNA 7500 and DNA 12000 Assay Protocol Starting the Chip Run 3 Click the Start button in the upper right of the window to start the chip run. The incoming raw signals are displayed in the Instrument context. 4 To enter sample information like sample names and comments, select the Data File link that is highlighted in blue or go to the Assay context and select the Chip Summary tab. Complete the sample name table . 5 To review the raw signal trace, return to the Instrument context.
Agilent DNA 7500 and DNA 12000 Assay Protocol Cleaning Electrodes after a DNA Chip Run 5 Cleaning Electrodes after a DNA Chip Run When the assay is complete, immediately remove the used chip from the Agilent 2100 bioanalyzer and dispose it according to good laboratory practice. After a chip run, perform the following procedure to ensure that the electrodes are clean (no residues are left over from the previous assay). NOTE CAUTION Use a new electrode cleaner with each new kit.
Agilent DNA 7500 and Checking Your Agilent DNA 7500 and DNA 12000 Assay Results DNA 7500 and DNA 12000 Ladder Well Results To check the results of your run, select the Gel or Electropherogram tab in the Data context. The electropherogram of the ladder well window should resemble those shown below.
Checking Your Agilent DNA 7500 and DNA 12000 Assay Results DNA 7500 and DNA 12000 Ladder Well Results Lower Marker Figure 2 6 Upper Marker DNA 12000 ladder Major features of a successful ladder run are: • 12 peaks for the DNA 7500 ladder and 13 peaks for the DNA 12000 ladder • All peaks are well resolved • Flat baseline • Correct identification of both markers If the electropherogram of the ladder well window does not resemble the one shown above, refer to the 2100 Expert Maintenance and Troubleshooti
6 Checking Your Agilent DNA 7500 and DNA 12000 Assay Results DNA 7500 and DNA 12000 Sample Well Results DNA 7500 and DNA 12000 Sample Well Results To review the results of a specific sample, select the sample name in the tree view and highlight the Results sub- tab. The electropherogram of the sample well window should resemble the one shown here.
Index Index Numerics assay menu, 17 ladder electropherogram, 20 loading gel-dye, 13 ladder, 15 marker, 14 samples, 15 B M base-plate, 7 marker, 14 C P chip priming station, 6, 7 chip selector, 8 cleaning electrodes, 19 cleaning up after assay, 19 preparation gel-dye, 11 protocol, 11 D results, 20, 22 running the DNA assay, 17 2100 expert software, 9, 16 A data context, 20 dye concentrate, 4, 11, 12 E electrodes, 16, 18, 19 electropherogram, 20 essential measurement practices, 10 F file pre
www.agilent.com In This Book you find the procedures to analyze DNA samples with the Agilent DNA 7500 and DNA 12000 reagent kit and the Agilent 2100 Bioanalyzer instrument. © Agilent Technologies, Deutschland GmbH 2000, 2000-2006, 2013 Printed in Germany 07/2013 *G2938-90024* *G2938-90024* G2938-90024 Rev.