Agilent Technologies MassCode PCR Research Solution - cDNA Synthesis, PCR, and PCR Purification Protocol Rev C User Guide
Summary
This comprehensive protocol manual guides researchers through advanced molecular biology techniques using MassCode kits. It details the complete workflow: synthesizing cDNA from complex nucleic acid samples, performing sensitive MassCode PCR amplification targeting specific sequences, and purifying the high-yield labeled products. Ideal for laboratory scientists and bioanalysts requiring dependable protocols for subsequent quantitative analysis via mass spectrometry.
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Agilent Mass Code PCR Research Solution c DNA Synthesis, PCR, and PCR Purification
Protocol Version C, April 2012 For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies
Page Summary Contents For Agilent Technologies MassCode PCR Research Solution - cDNA Synthesis, PCR, and PCR Purification Protocol Rev C User Guide
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Agilent Mass Code PCR Research Solution c DNA Synthesis, PCR, and PCR Purification Protocol Version C, April 2012 For Research Use Only. Not for use in diagnostic procedures. Agilent Technologies
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Notices © Agilent Technologies, Inc. 2011 Warranty Safety Notices No part of this manual may be reproduced in The material contained in this any form or by any means (including elec- document is provi...
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In this Guide... This document describes how to synthesize c DNA from a sample of nucleic acids, perform Mass Code PCR with the c DNA and purify the Mass Code tag labeled PCR products using the the Ma...
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c DNA Synthesis, PCR, and PCR Purification Protocol
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Contents Before You Begin 7 Overview 8 Purpose of the protocol 8 Protocol procedure 8 Important Information for Using the Protocol 10 Precautions to avoid RNase contamination 10 Contents of the kits 1...
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Contents Quick Reference Protocols 29 Synthesize first-strand c DNA 30 Amplify Mass Code targets by PCR 32 Purify the PCR products 33 c DNA Synthesis, PCR, and PCR Purification Protocol
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Mass Code c DNA Synthesis Kit and Mass Code PCR Reagent Kit Protocol Before You Begin Overview 8 Purpose of the protocol 8 Protocol procedure 8 Important Information for Using the Protocol 10 Precauti...
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Before You Begin Overview Overview Agilent’s Mass Code PCR Research Solution is a high- throughput platform for the detection of target nucleic acids based on multiplex PCR and LC/MS detection. An ove...
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Before You Begin Protocol procedure Figure 1 Overview of the Mass Code PCR Research Solution and the path for sample processing c DNA Synthesis, PC R, and PCR Purification Protocol
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Before You Begin Important Information for Using the Protocol Important Information for Using the Protocol Precautions to avoid RNase contamination Ribonucleases, such as RNase A, are very stable enzy...
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Before You Begin Contents of the kits Table 2 Contents of the Mass Code PCR Reagent Kit, Part Number 5190-3745 Mass Code 2× PCR Master Mix Kit, 192 Reactions, Box #5190-3564 Mass Code 2× PCR Master Mi...
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Before You Begin Storage conditions Storage conditions Mass Code c DNA Synthesis Kit Store all components at –20°C upon receipt. Mass Code PCR Reagent Kit Store the following boxes at –20°C upon recei...
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Before You Begin Required equipment and supplies Required equipment and supplies Table 5 Required Equipment and Supplies Description 1.5-ml microcentrifuge tubes Microcentrifuge Agilent Sure Cycler 88...
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Before You Begin Required equipment and supplies c DNA Synthesis, PCR, and PCR Purification Protocol
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Mass Code c DNA Synthesis Kit and Mass Code PCR Reagent Kit Protocol Procedures Synthesizing First-Strand c DNA 16 Prepare for the calibrator reactions 16 Dilute the MS2 phage (if needed) 17 Anneal pr...
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Procedures Synthesizing First-Strand c DNA Synthesizing First-Strand c DNA The c DNA synthesis step converts any RNA that is present in a nucleic acid mixture into first- strand c DNA. Prepare for the...
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Procedures Dilute the MS2 phage (if needed) Dilute the MS2 phage (if needed) If your nucleic acid samples already contain MS2 phage RNA, proceed to “Anneal primers to template” on page 17. If you did ...
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Procedures Anneal primers to template Table 6 Primer Annealing Reagent Mixture for Test Sample Reactions Volume per reaction Component If adding 4 μl nucleic acid If adding 10 μl nucleic acid Nucleic ...
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Procedures Perform reverse transcription Table 8 Primer Annealing Reagent Mixture for the CAL-IACRNA Reaction Volume per reaction Component If using MS2 nucleic acids If using whole MS2 phage MS2 phag...
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Procedures Perform reverse transcription Add 4.3 μl of the reverse transcription reagent mixture to each primer annealing reaction. Mix the reactions well (do not vortex). Incubate the reactions in th...
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Procedures Amplifying Mass Code Targets by PCR Amplifying Mass Code Targets by PCR The PCR amplification step amplifies target sequences using a multiplex primer mix. The primers in this mix are each ...
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Procedures Prepare the PCR reactions An example of a recommended plate setup is shown in Figure 2. The calibrator reactions are set up in row A of the plate. In this example, the target panel includes...
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Procedures Run the PCR program Add 4 μl of c DNA or PTC to each well. See “Design a PCR plate setup” on page 21 for guidelines. Run the PCR program Place the PCR plate in the Sure Cycler 8800. If you ...
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Procedures Purifying the PCR Products Purifying the PCR Products PCR purification removes excess primers, nucleotides, buffer components, and enzymes. It also removes any primer dimers that may have f...
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Procedures Purify the PCR products Purify the PCR products If you are not using all 96 wells of the binding plate, you can cover the unused portion of the NOTE plate with adhesive plate sealer and use...
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Procedures Purify the PCR products Wash the PCR products on the matrix Remove the plate sealer from the binding plate and add 500 μl of the prepared DNA Binding Solution to each well. This step is was...
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Procedures Purify the PCR products Remove the plate sealer from the binding plate and add 700 μl of 80% ethanol (see “Prepare the reagents” on page 24) to each well. This step is wash #3. Repeat the v...
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Procedures Place the plate in the autosampler Place the plate in the autosampler Place the autosampler plate in the front position of the LC/MS autosampler (2 positions for plates: one in front and on...
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Mass Code c DNA Synthesis Kit and Mass Code PCR Reagent Kit Protocol Quick Reference Protocols Synthesize first-strand c DNA 30 Amplify Mass Code targets by PCR 32 Purify the PCR products 33 This chap...
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Quick Reference Protocols Synthesize first-strand c DNA Synthesize first-strand c DNA If you will be adding MS2 phage to the c DNA synthesis reactions, prepare a 1:100 dilution of the provided stock. ...
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Quick Reference Protocols Synthesize first-strand c DNA For the CAL- IACRNA reaction, combine the components in Table 15. Table 15 Primer Annealing Reagent Mixture for the CAL-IACRNA Reaction Volume p...
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Quick Reference Protocols Amplify Mass Code targets by PCR Incubate the reactions in the Sure Cycler 8800 using the program listed in Table 17. Table 17 c DNA Synthesis Incubation Program Incubation S...
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Quick Reference Protocols Purify the PCR products Place the plate in the Sure Cycler 8800. For the respiratory target panel, run the program in Table 19. For a custom target panel, run a program suita...
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Quick Reference Protocols Purify the PCR products Perform the vacuum/centrifugation procedure on the binding plate according to the table below: Vacuum/Centrifugation Procedure for Immobilizing the PC...
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Quick Reference Protocols Purify the PCR products Perform the vacuum/centrifugation procedure on the binding plate according to the table below: Vacuum/Centrifugation Procedure for Washing the PCR Pro...
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Quick Reference Protocols Purify the PCR products Place the binding plate on top of a 96- well autosampler plate (provided). Check that the two plates are oriented in the same direction. Remove the pl...
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www.agilent.com In This Book This document describes how to synthesize c DNA from a sample of nucleic acids, perform Mass Code PCR with the c DNA and purify the Mass Code tag labeled PCR products usin...
Manual Details
| Brand | Agilent Technologies |
|---|---|
| Pages | 38 |
| File Size | 765.16 KB |
| Published | May 13, 2026 |
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Frequently Asked Questions
What is the primary function of this protocol?
The document describes synthesizing cDNA from nucleic acids, performing MassCode PCR, and purifying tagged products using specified kits.
How should purified PCR products be stored or used for analysis?
They can be placed directly into the LC/MS autosampler, or stored covered at 4°C (up to 1 week) or –20°C (long term).
What precautions should users take when working with this protocol?
Use MS-grade dH₂O for binding plates and consult the Quick Start Guide before starting an experiment.
Who should be contacted for technical product support?
Contact Agilent at (800) 227-9770 or send an email to [email protected].