Agilent MassCode PCR Research Solution
Contents
1. Honeywell Burdick amp Jackson Sigma LC MS Ultra CHROMASOLV Product number LC365 1 Product number 14263 1L Ammonium Sigma 7 5 M ammonium acetate solution Sigma ammonium acetate powder acetate Product number A2706 Product number 431311 Use the powder to create a 7 5 M stock solution Before the initial run and subsequent runs check the solvent volume levels to make sure you have enough for the run 1 Prepare a batch of 50 v v Methanol Water MS grade with a final concentration of 6 mM Ammonium Acetate a Rinse an empty bottle with MS grade water or use an empty bottle that contained MS grade water Set aside Do not wash the bottle with detergent b Using a micropipet add 0 8 mL of 7 5 M ammonium acetate solution directly to the bottom of a 500 mL graduated cylinder c Fill the graduated cylinder to 500 mL with MS grade water then pour the contents of the graduated cylinder into the empty bottle d Measure 500 mL of methanol and add to the bottle e Cap the bottle and mix by shaking gently Do not filter or sonicate the LC solvent LC MS Prep and Maintenance Guide Preparing the LC MS fora Run 1 2 Fill an empty clean 1 liter LC solvent bottle with the solvent Make sure that the LC solvent bottle contains 500 mL of this solution before each run Make sure you have updated the solvent levels in the MassCode PCR program See the Online Help gt How To Help gt Running Samples gt Working with the ChemSt2. by turning it counter clockwise In ChemStation increase the pump method s flow rate to 3 mL minute Do not save the method Continue purging until no more air bubbles appear in the line Decrease the pump method s flow rate to 0 8 mL minute then close the purge valve by turning it clockwise until snug do not overtighten Keep pumping for about 30 minutes The pressure measurement should not fluctuate more than about 1 Bar Put the LC MS into standby mode in one of two ways Through ChemStation e In the MassCode PCR Instrument menu select Enter instrument standby mode Close ChemStation in one of two ways e If MassCode PCR is not open exit ChemStation e Exit MassCode PCR then exit ChemStation LC MS Prep and Maintenance Guide Preparing the LC MS fora Run 1 Step 3 Prepare System Suitability vials Before running samples you must run the MS grade water that was used to elute the purified PCR samples to make sure it is free of background ions Vial 1 and a calibrator to ensure the MS measurements are accurate Vial 2 The System Suitability methods using these samples check mass assignment MS calibration and reagent contamination and prepare the system for data analysis 1 Before each run make sure Vial 1 the glass vial contains no less than 250 uL of the MS quality water used to purify the samples Do not cap the vial 2 Prepare the calibrator sample Vial 2 a Add 950 uL of MS grade wa
3. inlet to see if solvent is flowing If the flow is OK check sample location and or injection V Is the unknown in the specified location If not add it to the specified well If there is no signal after making sure the unknown is in the right location see If there is no sample signal test MS on page 22 If MS Checktune and or Autotune passes contact Agilent support to make sure the autosampler is working LC MS Prep and Maintenance Guide Troubleshooting the LC MS 3 If pump pressure fluctuates more than expected V Make sure the purge valve is closed V Check the MS inlet filter to see if it needs replacing V Make sure the LC flow is being delivered properly Measure the flow rate using a graduated cylinder and timer 0 8 mL minute If the UV LED does not turn green After the UV module is turned on it takes 10 to 30 minutes for it to stabilize and the LED to turn green If it has not turned green after 30 minutes the lamp may require replacement If the UV LED flashes red and amber After the UV module is turned on it performs internal self diagnostics to verify that it can successfully illuminate the UV lamp If the UV lamp is not bright enough or if the UV tray is not properly installed the UV LED will flash between red and amber In this case you may need to remove and reinstall the UV tray and lamp If the UV tray tubing is significantly discolored or has been used for more than 1200 hours it
4. may require replacement If the UV lamp has been used for more than 3600 hours it may require replacement Use LC diagnostic tools Use Lab Advisor diagnosis tools LC to troubleshoot LC problems Click Instrument gt Launch Lab Advisor LC MS Prep and Maintenance Guide 21 3 Troubleshooting the LC MS Diagnosing and Resolving MS Problems 22 If there is no sample signal test MS You can test the MS before testing the LC when there is no sample signal or vice versa See If there is no sample signal test LC on page 20 Sometimes it is easier to test for LC problems first After testing the LC flow and sample location and injection test for MS issues V Is the flow going from the MS inlet into the MS nebulizer Bypass the MS inlet selection valve assembly and plumb the LC outlet directly to the MS nebulizer Y If yes does the MS pass Checktune Before doing a Checktune reconnect the MS nebulizer to the selection valve If no check the calibrant level Refill calibrant And redo Checktune Y If yes does the MS pass Autotune If no contact Agilent support See Agilent Tech Support information on page 4 If the MS LED turns red A red light could indicate a number of problems Only the ones below you can correct If the LED stays red then contact Agilent support V Check the nitrogen flow Replace the nitrogen tank if applicable V Check for leaks in the LC or MS V Check that the spra
5. red and amber 21 Use LC diagnostic tools 21 Diagnosing and Resolving MS Problems 22 If there is no sample signal test MS 22 Ifthe MS LED turns red 22 Use MS diagnostic tools 23 LC MS Prep and Maintenance Guide LC MS Prep and Maintenance Guide Agilent MassCode PCR Research Solution LC MS Prep and Maintenance Guide 1 Preparing the LC MS for a Run Step 1 Prepare the LC Solvent 8 Step 2 Prime and equilibrate the LC 10 Step 3 Prepare System Suitability vials 11 Step 4 Place System Suitability vials in the Autosampler 12 After installation of the Agilent MassCode PCR hardware and software prepare the LC MS hardware for a run by making up the solvent priming and equilibrating the system and preparing the solutions for System Suitability calibration runs If you need help with the instructions see Agilent Tech Support information on page 4 wie Agilent Technologies 7 1 Preparing the LC MS for a Run Step 1 Prepare the LC Solvent Agilent recommends specific sources for the reagents needed to prepare this solvent Table 1 provides the ordering information for the recommended source If the recommended source is unavailable use the alternative source in its place Table 1 Recommended sources for LC solvent reagents Reagent Recommended Source Alternative Source Methanol Honeywell Burdick amp Jackson Sigma LC MS Ultra CHROMASOLV Product numbers LC230 1 and GC230 2 5 Product number 14262 1L Water
6. 00 Series Single Quad LC MS System Maintenance Guide G1960 90054 If you need to call Agilent Support see Agilent Tech Support information on page 4 2 Maintaining the LC MS HPLC Modules Solvent Delivery System Solvent bottle filter Check and clean the filter in the solvent bottle exchange if necessary See Chapter 9 Maintenance of the Agilent 1260 Infinity Isocratic Pump and Quaternary Pump User Manual G1310 90012 Pump For instructions on scheduled maintenance tasks for the pump see Chapter 9 Maintenance of the Agilent 1260 Infinity Isocratic Pump and Quaternary Pump User Manual G1310 90012 If the pump runs dry see p 59 of the manual for instructions on Initial Priming of the pump Initial priming is a method to initiate solvent flow through an empty line and eliminate air bubbles from the flow Autosampler For instructions on scheduled maintenance tasks for the autosampler see Chapter 10 Maintenance of the Agilent 1260 Infinity High Performance Autosampler User Manual G1367 90012 14 LC MS Prep and Maintenance Guide Maintainingthe LC MS 2 MassCode UV Module For instructions on scheduled maintenance tasks for the UV module see Chapter 4 Maintenance of the Agilent MassCode UV Module User Manual G5201 90000 After 50 days or 1200 hours Exchange tray 150 days about 5 months 3600 hours Exchange lamp LC MS Prep and Maintenance Guide 15 2 Maintaining the LC MS
7. Agilent MassCode PCR Research Solution LC MS Prep and Maintenance Guide Revision C July 2012 For Research Use Only Not for use in diagnostic procedures ote Agilent Technologies Notices Agilent Technologies Inc 2012 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 G9200 90006 Edition Version C July 2012 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Technical Support For technical product support contact Agilent at 800 227 9770 or masscode support agilent com Limited Use License for End Users For Research Use Only Not for use in diag nostic procedures Warranty The material contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damage
8. ChemStation b In the MSD Tune view under the Instrument menu select Turn Off Spray Chamber It takes about 15 minutes for the ion source to cool 2 Make sure the ion source is cool and then wipe it out with a lint free cloth and methanol 3 Inspect corona needle and nebulizer and clean if necessary Biannually yearly or as needed As needed depending on the pressure profile that the pump generates e g sudden increase in pressure exchange the inlet frit filter for the MS Agilent recommends this be done after running 20 plates Every 6 months change the oil in the rough pump If you have a service contract service engineers from Agilent will do this Yearly replace the rotor seal in the selection valve This valve directs the flow to the nebulizer or waste from the calibrant or LC Yearly replace the nitrogen filter that sits in between the nitrogen source and MS Recommended nitrogen source when no in house nitrogen ultra high purity liquid nitrogen Dewar LC MS Prep and Maintenance Guide 17 2 Maintaining the LC MS Biannually yearly or as needed 18 LC MS Prep and Maintenance Guide Agilent MassCode PCR Research Solution e LC MS Prep and Maintenance Guide ee 3 Troubleshooting the LC MS e a e g Diagnosing and Resolving LC Problems 20 If there is no sample signal test LC 20 If pump pressure fluctuates more than expected 21 If the UV LED does not turn green 21 If the UV L
9. ED flashes red and amber 21 Use LC diagnostic tools 21 Diagnosing and Resolving MS Problems 22 If there is no sample signal test MS 22 Ifthe MS LED turns red 22 Use MS diagnostic tools 23 This chapter helps you troubleshoot the LC MS for easy to resolve problems and lets you know when to call Agilent support If you need to call Agilent support see Agilent Tech Support information on page 4 pee Agilent Technologies 19 3 Troubleshooting the LC MS Diagnosing and Resolving LC Problems 20 If there is no sample signal test LC You can test the LC before testing the MS when there is no sample signal or vice versa If there is still no signal after confirming proper LC performance see If there is no sample signal test MS on page 22 Or contact Agilent support See Agilent Tech Support information on page 4 Of course under these conditions System Suitability will not pass its error message may give a clue about the cause of the problem See Troubleshooting Help in the Online Help for a list of System Suitability error messages Check these situations in this order Y Is there sufficient HPLC solvent Make up a new batch See Step 1 Prepare the LC Solvent on page 8 Be sure to always check the solvent level before running a plate V Is the pump purge valve closed Close the purge valve V Is the LC flow going through the UV module Disconnect the LC tubing from the MS
10. Single Quad LC MS System CAUTION For instructions on how to do these scheduled maintenance tasks for the MS refer to the Agilent 6100 Series Single Quad LC MS Systems Maintenance Guide G1960 90054 Do NOT run Checktune daily as recommended in the Single Quad LC MS Systems Maintenance Guide Run it only when indicated by a System Suitability failure Do NOT run Autotune monthly Run it only when Checktune fails after the System Suitability results have indicated a Checktune is necessary Daily 16 Put MS in standby or have it done automatically at the end of the run default setting See Online Help gt How To Help gt Running Samples gt Working with ChemStation Advanced Standby e Turns down the drying gas flow to 3 L minute e Turns down the nebulizer pressure to 15 psi e Turns down the drying gas temperature to 300 degrees e Turns down the vaporizer temperature to 200 degrees e Turns off the pump e UV does not change during standby the lamp stays on LC MS Prep and Maintenance Guide Maintainingthe LC MS 2 Weekly lon Source and Nebulizer Cleaning the ion source and nebulizer is best done at the end of a day when no more runs will be made because the background increases temporarily 1 Turn temperatures off first There is no need to put the MS into standby unless LC flow is running To turn off the ion source a Under the Instrument menu in the MassCode PCR software select Show
11. ard 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 LC MS Prep and Maintenance Guide In this Guide LC MS Prep and Maintenance Guide This document describes all the post installation tasks for the LC MS system preparing the LC MS for a run maintaining the LC MS and diagnosing and resolving LC and MS problems Consult the Quick Start Guide for an overview of preparing the LC MS for a run and a description of how the information in this guide fits into the overall Agilent MassCode PCR Research Solution workflow Preparing the LC MS for a Run After installation of the Agilent MassCode PCR hardware and software prepare the LC MS hardware for a run by making up the solvent priming and equilibrating the system and preparing the solutions for System Suitability calibration runs Maintaining the LC MS The recommendations and instructions for maintenance tasks in this chapter include only those for essential maintenance on a regular basis Troubleshooting the LC MS This chapter helps you troubleshoot the LC MS for easy to resolve problems and lets you know when to call Agilent support Before you start Where to find more information The Quick Start Guide provides an overview of the Agilent MassCod
12. ation gt Update Solvent Levels Prepare a new batch of solvent as needed or every week Do not use the solvent for longer than 7 days If left too long exposed to air CO4 the pH of the solvent can become acidic and MassCode tagged molecules can accumulate in the LC signals go down noise goes up Or if the pH becomes alkaline more unwanted photolysis products are generated Ideally with little exposure to air and the presence of ammonium acetate the pH should be around 7 If the pH is not between 6 8 replace the solvent LC MS Prep and Maintenance Guide 1 10 Preparing the LC MS for a Run Step 2 Prime and equilibrate the LC If you are changing from a different solvent to the MassCode PCR solvent you must purge the system of air bubbles priming and ensure the pressure is stable equilibrating If you are refilling MassCode PCR solvent to continue running well plates skip this Step 1 Open ChemStation in one of two ways e Double click the ChemStation icon on your desktop e Through the MassCode PCR software a Double click the MassCode PCR Online icon b In the Instrument menu select Show ChemStation If the instrument is in standby or off turn the instrument on in one of two ways e Through ChemStation e In the MassCode PCR Instrument menu select Turn Instrument On Make sure the MS stream selector valve is set to waste default On the G1310A B Isocratic Pump open the pump purge valve black knob
13. e PCR Research Solution The Installation Guide covers installation of the MassCode PCR software Install the software before you start the exercises in this Familiarization Guide The software s online help system provides in depth information on the operation of the MassCode PCR program and can be displayed in the following ways e Click Help gt Contents from the menu bar e Press F1 to get more information about the currently displayed screen or dialog box Agilent Tech Support information E mail masscode support agilent com or click Help gt Email Feedback in the software Phone From the US and Canada call 800 227 9770 select options 3 4 3 For Agilent s worldwide sales and support center telephone numbers go to www agilent com chem contactus LC MS Prep and Maintenance Guide Contents 3 Preparing the LC MS foraRun 7 Step 1 Prepare the LC Solvent 8 Step 2 Prime and equilibrate the LC 10 Step 3 Prepare System Suitability vials 11 Step 4 Place System Suitability vials inthe Autosampler 12 Maintaining the LC MS 13 HPLC Modules 14 Solvent Delivery System 14 Autosampler 14 MassCode UV Module 15 Single Quad LC MS System 16 Daily 16 Weekly 17 Biannually yearly or as needed 17 Troubleshooting the LC MS 19 Diagnosing and Resolving LC Problems 20 If there is no sample signal test LC 20 If pump pressure fluctuates more than expected 21 If the UV LED does not turn green 21 If the UV LED flashes
14. rence Help Online Help for information on the calculations for the System Suitability measurements You are now ready to prepare the well plate for a run For instructions for the Respiratory Pathogen Panel see the Respiratory Sample Preparation Protocol and the cDNA Synthesis PCR and PCR Purification Protocol If you have already prepared and stored a well plate and are ready to run it follow these instructions for placing the well plate in the autosampler of the LC MS e Place the well plate in the front position of the autosampler There are 2 positions for plates one in front and one in back e Place the plate so that Row A is in the back and Row H faces the front If you want to turn off the light in the autosampler see the Online Help The default setting is On LC MS Prep and Maintenance Guide Agilent MassCode PCR Research Solution LC MS Prep and Maintenance Guide ee 2 Maintaining the LC MS HPLC Modules 14 Solvent Delivery System 14 Autosampler 14 MassCode UV Module 15 Single Quad LC MS System 16 Daily 16 Weekly 17 Biannually yearly or as needed 17 The recommendations and instructions for maintenance tasks in this chapter include only those for essential maintenance on a regular basis For more sophisticated maintenance tasks see e Agilent 1260 Infinity Series LC Module User Manuals Maintenance chapters e Agilent MassCode UV Module User Manual Maintenance chapter G5201 90000 e Agilent 61
15. s in con nection with the furnishing use or performance 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 doc ument that conflict with these terms the warranty terms in the separate 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 U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation 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 haz
16. ter to a tube containing 50 uL of thawed System Suitability calibrator Box p n 5190 3743 in the PCR reagent kit p n 5190 3745 b Transfer the solution to a polypropylene autosampler vial do NOT use a glass vial Do not cap the vial You can purchase glass vials and polypropylene autosampler vials from Agilent Glass vials part number 5182 0716 100 vials package Polypropylene autosampler vials part number 5182 0567 100 vials package 3 Before each run ensure Vial 2 contains no less than 250 uL of calibrator Do not use the System Suitability calibrator for longer than 7 days even if there is material left for more injections The System Suitability calibrator lasts for 4 to 5 runs Total volume is 1000 uL after dilution the autosampler injects 150 uL and 250 uL must be present in the vial to ensure that enough is available for the injections The kit contains one vial of System Suitability calibrator per plate LC MS Prep and Maintenance Guide 11 1 12 Preparing the LC MS for a Run Step 4 Place System Suitability vials in the Autosampler The autosampler contains 10 vial positions to the right of the well plate locations front and back e Place the vial containing MS grade water in vial position 1 e Place the vial containing calibrator from the PCR reagent kit prepared according to instructions in step 2 of Step 3 Prepare System Suitability vials on page 11 in vial position 2 See the Refe
17. y chamber is not open not normally an issue An error also appears in ChemStation See Use MS diagnostic tools on page 23 LC MS Prep and Maintenance Guide Troubleshooting the LC MS 3 Use MS diagnostic tools Use ChemStation diagnostic tools MS to troubleshoot problems with the mass spectrometer 1 Click Instrument gt Show ChemStation Diagnosis The ChemStation Method and Run Control View appears 2 Use the ChemStation Diagnosis Toolbar near the top of the screen to diagnose the MS problem Test Results Logbooks ce wl 3j OQ PY Analytic Observed Symptoms v Possible Causes LC MS Prep and Maintenance Guide 23 3 Troubleshooting the LC MS Use MS diagnostic tools 24 LC MS Prep and Maintenance Guide www agilent com In This Book This document describes all the post installation tasks for the LC MS system preparing the LC MS for a run maintaining the LC MS and diagnosing and resolving LC and MS problems Agilent Technologies Inc 2012 Version C July 2012 G9200 90006 Ss Agilent Technologies
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