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Agilent DNA 1000 Kit Guide

1. Agilent DNA 1000 Kit Guide XE Agilent Technologies Notices O Agilent Technologies Inc 2000 2000 2006 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G2938 90014 Edition 08 2006 Printed in Germany Agilent Technologies Hewlett Packard Stra se 8 76337 Waldbronn Germany s Caliper amp LabChip and the Lab Chip logo are registered trade SLAE marks of Caliper Technologies Corp in the U S and other countries Adobe and Acrobat are U S registered trademarks of Adobe Systems Incorporated Microsoft is a U S registered trademark of Microsoft Corporation Software Revision This guide is valid for B 01 02 and B 02 02 and higher revisions of the Agilent Expert software where 02 refers to minor revisions of the software that do not affect the techni cal accuracy of this guide Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including b
2. 3 Setting up the Assay Equipment and 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 MS2 S8 MS2 S9 To set up the vortex mixer adjust the speed knob to 2400 rpm 8 Agilent DNA 1000 Setting up the Assay Equipment and Bioanalyzer 3 Starting the 2100 Expert Software Starting the 2100 Expert Software To start the software 1 Go to your desktop and double click the following icon sd The screen of the software appears in the Instrument context The icon in the upper part of the screen represents the current instrument PC communication status iB Lid closed no chip or m chip empty S Eig v Lid open aim Pi COL REED pare liess Dimmed icon no communication i T minia E TI I i BELL Ll i umma juge puri ee qi mam Lid closed chip inserted DNA or demo assay selected 2 If more than one instrument is connected to your PC select the instrument you want to use in the tree view e All Instruments Y DE13 701065 d y DEOZ DD 386 Agilent DNA 1000 9 Agilent DNA 1000 o e e e e e 00 ee Essential Measurement Practices e e e Handle and store
3. 0354 Physical Specifications Analytical Specifications Type Specification Type Agilent DNA 1000 Assay Analysis run time 35 minutes Sizing range 25 1000 bp Number of samples 12 samples chip Typical sizing resolution 5 bp 25 100 bp 5 100 500 bp 10 500 1000 bp sample volume 1 ul Sizing accuracy 10 for ladder as sample Kit stability 4 months Storage temperature Sizing reproducability 5 CV for ladder as sample see individual box Quantitation accuracy 20 CV for ladder as sample Quant reproducibility 25 500 bp 15 CV 500 1000 bp 5 CV for ladder as sample Quantitative range 0 1 50 ng yl Maximum salt 250 mM for KCI or NaCl 15 mM for MgCl Some fragments below 70 bp may deviate from the above specifications 4 UE Agilent Technologies Agilent DNA 1000 ove ep eece Equipment Required for a DNA 1000 r Assay e Equipment Supplied with the Agilent 2100 Bioanalyzer Chip priming station reorder number 5065 4401 KA vortex mixer Additional Material Required Not Supplied Pipettes 10 ul 100 ul and 1000 ul with compatible tips 0 5 ml microcentrifuge tubes for sample preparation Microcentrifuge Check the Agilent Lab on a Chip webpage for details on assays www agilent com chem labonachip RE Agilent Technologies Agilent DNA 1000 o e ec Setting up the Assay Equipment and eu Bioanalyzer Before beginning the chip preparation protocol e
4. Well Results 20 1000 Sample Well Results 22 Index 23 Agilent DNA 1000 Agilent DNA 1000 o t 00 ee Agilent DNA 1000 Kit amp e e e e Agilent DNA 1000 Kit reorder number 5067 1504 DNA Chips DNA 1000 Reagents reorder number 5067 1505 25 DNA Chips yellow DNA 1000 Ladder 1 Electrode Cleaner 9 green DNA 1000 Markers 15 1500 bp 2 vials Syringe Kit blue DNA Dye Concentrate 1 vial 1 Syringe red DNA Gel Matrix 3 vials 3 Spin Filters This product is provided under an agreement between Molecular Probes Inc a wholly owned subsidiary of Invitrogen Corporation and Agilent Technologies The manufacture use sale or import of this product may be subject to one or more of U S patents pending applications and corresponding international equivalents owned by Molecular Probes Inc The purchaser has the non transferable right to use the product to detect protein and or nucleic acids in microfluidics analysis systems for one or more of the subfields of research development quality control forensics environmental analysis biodefense food safety testing veterinary diagnostics or human diagnostics according to use indicated on the product label or accompanying product literature For information on obtaining a license contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 9132 Tel 541 465 8300 Fax 541 335
5. 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 when not in use Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use Protect dye and dye mixtures from light Remove light covers only when pipetting The dye decomposes when exposed to light and this reduces the signal intensity e Always insert the pipette tip to the bottom of the well when dispensing the liquid Placing the pipette at the edge of the well may lead to poor results Use a new syringe and electrode cleaners with each new kit Use loaded chips within 5 minutes after preparation Reagents might evaporate leading to poor results Do not touch the Agilent 2100 bioanalyzer during analysis and never place it on a vibrating surface 10 RES Agilent Technologies Agilent DNA 1000 o e t ee ec Agilent DNA 1000 Assay Protocol e o o e e e 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 WARNING Handling DMSO Kit components contain DMSO Because the dye binds to nuclei
6. expertiDatalz005 11 22 File Prefix 2100 expert max 16 characters Data Acquisition Parameters Run sample 1 to 12 Agilent DNA 1000 17 5 Agilent DNA 1000 Assay Protocol CAUTION 18 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 3 Start 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 Sample SampleComment Observation Result Label Result Color E BoBme2 201 2 pp samples m 5 Samples oo o amp o o samples 0 00 0 2 10 Sample 10 0 T 2 28012 Chip Comments Sample Information Study Information Import Export 5 To review the raw signal trace return to the Instrument context 6 After the chip run is finished remove the chip from the receptacle of the bioanalyzer and dispose it according to good laboratory practices Contamination of electrodes Leaving the chip for a period longer than 1 hour e g over night in the bioanalyzer may cause contamination of the electrodes Immediately remove the chip after a run Agilent DNA 1000
7. 000 Assay eu e 6 DNA 1000 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 to those shown below Lower Marker Figure 1 DNA 1000 ladder 20 E Agilent Technologies Agilent DNA 1000 Checking Your Agilent DNA 1000 Assay Results 6 Major features of a successful ladder run are 13 peaks for DNA 1000 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 Troubleshooting Guide for assistance 21 6 Checking Your Agilent DNA 1000 Assay Results DNA 1000 Sample Well Results 22 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 Upper zl Lower Marker Mtarker H i I AM dbi ee S 40 5 50 70 80 an 00 110 ix Figure 2 DNA peaks of a successful sample run Major features for a successful DNA sample run are All sample peaks appear between the lower and upper marker peaks If some sample peaks are outside the marker bracket adjust the upper or lower marker Please refer to the 2100 Expert User s Guide or Online Help for d
8. Agilent DNA 1000 Assay Protocol 5 Cleaning Electrodes after a DNA 1000 Chip Run CAUTION Agilent DNA 1000 When the assay is complete immediately remove the used chip from the Agilent 2100 bioanalyzer and dispose it according to good laboratory practice Then perform the following procedure to ensure that the electrodes are clean i e no residues are left over from the previous assay Use a new electrode cleaner with each new kit Leak currents between electrodes Liquid spill might cause leak currents between the electrodes Never fill too much water in the electrode cleaner 1 Slowly fill one of the wells of the electrode cleaner with 350 ul deionized analysis grade water Open the lid and place electrode cleaner in the Agilent 2100 bioanalyzer Close the lid and leave it closed for about 10 seconds Open the lid and remove the electrode cleaner cC N Wait another 10 seconds to allow the water on the electrodes to evaporate before closing the lid After 5 assays empty and refill the electrode cleaner After 25 assays replace the used electrode cleaner by a new one When switching between different assays a more thorough cleaning may be required Refer to the maintenance chapter on this CD Maintenance and Troubleshooting Guide for details which is part of the Online Help of the 2100 bioanalyzer software 19 Agilent DNA 1000 Results ns e E e e Checking Your Agilent DNA 1
9. Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met Agilent DNA 1000 Contents 1 Agilent DNA 1000 Kit 4 2 Equipment Required for a 1000 Assay 5 J Setting up the Assay Equipment and Bioanalyzer 6 Setting up the Chip Priming Station 7 Setting up the Bioanalyzer 8 Vortex Mixer 8 Starting the 2100 Expert Software 9 4 Essential Measurement Practices 10 5 Agilent DNA 1000 Assay Protocol 11 Preparing the Gel Dye 11 Loading the Gel Dye Mix 13 Loading the Marker 14 Loading the Ladder and the Samples 15 Inserting a Chip in the Agilent 2100 Bioanalyzer 16 Starting the Chip 17 Cleaning Electrodes after a 1000 Chip 19 6 Checking Your Agilent DNA 1000 Assay Results 20 DNA 1000 Ladder
10. c acids it should be treated as a potential mutagen and used with appropriate care Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples Handle the DMSO stock solutions with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues 1 Allow the DNA dye concentrate blue 9 and DNA gel matrix red 9 to equilibrate to room temperature for 30 minutes It is important that all the reagents have room temperature before starting the next step Protect the dye concentrate from light RE Agilent Technologies 11 5b Agilent DNA 1000 Assay Protocol 2 Vortex the blue capped DNA dye concentrate blue 9 for 10 seconds and spin down Make gel dye mix sure the DMSO is completely thawed 3 Pipette 25 ul of the blue capped dye 25 pl dye concentrate blue 9 into a red capped DNA gel matrix vial red 9 Store the dye concentrate at 4 in the dark again Always use the volumes indicated Using different volumes in the same ratio will produce inaccurate results 4 Cap the tube vortex for 10 seconds Visually inspect proper mixing of gel and dye 5 Transfer the gel dye mix to the top receptacle of a spin filter 6 Place the spin filter in a microcentrifuge and spin for 15 minutes at room temperature at 2240 g 20 for Eppendorf microcentrifuge this corresponds to 6000 rpm 1 Discard the filter according to good laboratory pra
11. ctices Label the tube and include the date of preparation The prepared gel dye mix is sufficient for 10 chips Use the gel dye within 4 weeks of preparation Protect the gel dye mix from light Store the gel dye mix at 4 when not in use for more than 1 hour 12 Agilent DNA 1000 Agilent DNA 1000 Assay Protocol 5 Loading the Gel Dye Mix 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 lowest 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 2 Take anew DNA chip out of its sealed bag and place the chip on the chip priming station J Pipette 9 0 ul of the gel dye mix at the bottom of the well marked When pipetting the gel dye mix make sure not to draw up particles that may sit at the bottom of the gel dye mix vial Insert the tip of the pipette to the bottom of the chip well when dispensing This prevents a large air bubble forming under the gel dye mix Placing the pipette at the edge of the well may lead to poor results 4 Setthe timer to 60 seconds make sure that the plunger is positioned at 1 ml and then close the chip priming station The lock of the latch will click when the Priming Station is closed correctly Agilent DNA 1000 13 5 Ag
12. e lid The electrodes in the cartridge fit into the wells of the chip 5 The 2100 expert software screen shows that you have inserted a chip and closed the lid by displaying the chip icon at the top left of Instrument context 08 Tuum 201600107 AID ba Ci abeam LOB arl DEOR 11 28 SHE ET j Bahet Fi mpm nl ee aar Bis 2 af fa Tur ewige rnb cadi PITE a ar nom cd on rs aas af tha and Pa med unas Tee af all recante et IIT Turm Agilent DNA 1000 Agilent DNA 1000 Assay Protocol 5 Starting the Chip Run 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 2 Accept the current File Prefix or modify it Data will be saved automatically to a file with a name using the prefix you have just entered At this time you can also customize the file storage location and the number of samples that will be analyzed Destination Default Cii alyzeriz100 2005 11 22 Custom C alyzeri2100
13. etails e Flat baseline e Baseline readings at least 5 fluorescence units see Zero Baseline in the User s guide or Online Help for details of how to see the baseline readings Marker readings at least 3 fluorescence units higher than baseline readings e Both marker peaks well resolved from sample peaks depends on sample Agilent DNA 1000 Index Numerics 2100 expert software 9 17 chip selector 6 8 D data context 20 dye concentrate 4 E electrode cleaning 19 electrodes 16 19 electropherogram 20 22 essential measurement practices 10 F file prefix 17 G gel dye 11 13 14 instrument context 16 17 L ladder 15 20 ladder electropherogram 20 M marker 14 Agilent DNA 1000 P preparation gel dye 11 protocol 11 R results 20 S sample electropherogram 22 samples 15 set up base plate bioanalyzer 8 chip priming station 7 chip selector 8 syringe clip 7 specifications analytical 4 physical 4 syringe 4 7 syringe clip 7 V vortexer 8 Index 23 www agilent com In This Book you find the procedures to analyze DNA samples with the Agilent DNA 1000 reagent kit and the Agilent 2100 expert bioanalyzer Agilent Technologies Deutschland GmbH 2000 2000 2006 Printed in Germany 08 2006 014 RES Agilent Technologies 62938 90
14. ilent DNA 1000 Assay Protocol 5 Press the plunger of the syringe down until it is held by the clip dion OOOO 6 Wait for exactly 60 seconds and then release the plunger with the clip release mechanism OROKOR 7 Visually inspect that the plunger moves back at least AAR 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 0 ul of the gel dye mix in each of the wells marked OOOO OOOO 9 ul gel dye Protect the gel dye mix from light Store the gel dye mix at 4 C when not in use for more than 1 hour Loading the Marker 1 Pipette 5 ul of green capped DNA marker green 9 into the well marked with the ladder symbol 4 and into each of the 12 sample wells bod p T EX LJ M NO 5 pl marker Do not leave any wells empty or the chip will not run properly Add 5 ul of green capped DNA marker green 9 plus 1 ul of deionized water to each unused sample well 14 Agilent DNA 1000 Agilent DNA 1000 Assay Protocol Loading the Ladder and the Samples CAUTION Agilent DNA 1000 1 Pipette 1 ul of the yellow capped DNA ladder yellow in the well marked with the ladder symbol 4 beds OOOO OOOO O00 1 pl ladder 2 Ineach of the 12 sample wells pipette 1 ul of sample used wells or 1 ul of deionized water unused Pe wells eooo LJ E EE NO 1 pl sample Wrong vortexing speed If vortexi
15. ng speed is too high liquid spill that disturbs the analysis may occur for samples generated with detergent containing PCR buffers Reduce vortexing speed to 2000 rpm For optimal results samples should be of pH 6 to 9 and should not have an ionic content greater than twice that of a typical PCR buffer 3 Set the timer to 60 seconds 5 4 Place the chip horizontally in the adapter of the IKA vortex mixer and make sure not to damage the buldge that fixes the chip during vortexing 5 Vortex for 60 seconds at 2400 rpm 6 Refer to the next topic on how to insert the chip in the Agilent 2100 bioanalyzer Make sure that the run is started within 5 minutes 15 5 Agilent DNA 1000 Assay Protocol Inserting a Chip am th Fy A ron mmt 7 ann D P Ar tl 1e AGI lent 2100 Bioanal 2 Inserting a Chip in the Agilent 2100 Bioanalyzer CAUTION 16 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 Sensitive electrodes and liquid spills Forced closing of the lid may damage the electrodes and dropping the lid may cause liquid spills resulting in bad results Do not use force to close the lid and do not drop the lid onto the inserted chip 4 Carefully close th
16. nsure that the chip priming station and the bioanalyzer are set up and ready to use You have to replace the syringe at the chip priming station with each new DNA kit e adjust the base plate of the chip priming station adjust the syringe clip at the chip priming station e adjust the bioanalyzer s chip selector set up the vortex mixer finally make sure that you start the software before you load the chip fheAgilent DNA 1000 assay is a high sensitivity assay Please read this guide carefully and follow all instructions to guarantee satisfactory results 6 EE Agilent Technologies Setting up the Assay Equipment and Bioanalyzer 3 Setting up the Chip Priming Station 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 2 Adjust the base plate a Open the chip priming station by pulling the latch b Using a screwdriver open the screw at the underside of the base plate c Lift the base plate and insert it again in position C Retighten the screw J Adjust the syringe clip a Release the lever of the clip and slide it down to the lowest position Agilent DNA 1000 7
17. ut not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 orasa commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or disclo sure of software is subject to Agilent Tech nologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited

Agilent DNA 1000 Kit Guide

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