Product Class: Other

Multiplex PCR 5X Master Mix
cloned at NEB recombinant No pcr 5

Product Introduction

Multiplex with flexibility and minimal optimization

  • Simultaneously detect two or more products in a single reaction
  • Enhanced buffer formulation for optimal performance 
  • 5X master mix format for easy reaction setup - just add template DNA and primers
Catalog # Size Concentration
M0284S 100 reactions (50.0 µl) 5 X

Product Information

Description

Multiplex PCR can simultaneously detect two or more products in a single reaction. There is an increasing demand for multiplex PCR techniques in assays conducted in research laboratories and forensic/diagnostic genotyping assays (1,2). Multiplex PCR can also be used for semi-quantitative gene expression analysis using cDNA templates. The NEB Multiplex PCR 5X Master Mix is an easy-to-use solution featuring high quality recombinant Taq DNA Polymerase. The mix is optimized for high yield and robust performance. Its performance is illustrated in a 15-plex PCR using human genomic DNA (Figure 1) and an 8-plex PCR using cDNA products as templates (Figure 2). The 5X formulation allows the user maximal input of primers, template DNAs and additional components.

Figure 1
Figure 1:
15-plex PCR using varying amounts of human genomic DNA. 1X Multiplex PCR 5X Master Mix was used with 0.15 μM of each primer. The cycling conditions were 95°C for 1 minute, 35 cycles of 95°C for 20 seconds, 60°C for 1 minute and 68°C for 2 minutes. Marker M is the 1 kb Plus DNA Ladder (NEB #N3200).
Figure 2
Figure 2
8-plex PCR using cDNA products from 1 ng human spleen total RNA. Cycling conditions were 95°C for 1 minute, 30 cycles of 95°C for 20 seconds, 60°C for 30 seconds and 68°C for 2 minutes. Amplicon lengths are indicated next to gel.
Figure 3
Figure 3:
Comparison of PCR product yields between single-plex and multiplex PCR reactions. Figure A shows amplification of human genomic DNA fragments (sizes are listed to the left of the gel). Lane 1 is a 3-plex reaction, Lanes 2-4 are single-plex reactions, and Lanes 5–6 are 2-plex reactions; Lane 7 is the 1 kb DNA Ladder (NEB #N3232 ). Figure B shows the results of expression analysis of five mRNAs using cDNA templates (first strand synthesis was carried out using the ProtoScript Kit with human spleen total RNA); Lane 1 is a 5-plex PCR from cDNA reactions, while Lanes 2–6 are single-plex PCR reactions; Lane 7 is the 100 bp DNA Ladder (NEB #N3231).

Product Source

An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1.
This product is related to the following categories:
Master Mixes,
Taq DNA Polymerase,
This product can be used in the following applications:
Multiplex PCR,
Specialty PCR,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

1X Master Mix Composition

20 mM Tris-HCl
50 mM KCl
30 mM NH4Cl
2.5 mM MgCl2
0.3 mM dNTPs
3.2% Glycerol
0.08% IGEPAL® CA-630
0.07% Tween® 20
67 units/ml Taq DNA Polymerase
pH 8.9 @ 25°C

Heat Inactivation

No

Molecular Weight

Theoretical: 94000 daltons

5' - 3' Exonuclease

Yes

3' - 5' Exonuclease

No

Strand Displacement

No

Resulting Ends

  • Single-base 3´ Overhangs

Unit Assay Conditions

1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 15 nM primed M13 DNA.

Unit Definition:
One unit of Taq DNA Polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.

Features

  • Multiplex with Taq

Product Notes

  1. Use high quality primers (desalted or HPLC purified).
  2. Accurately quantify and adjust primer concentrations to 50 μM in 0.5X TE Buffer.
  3. Individually test the PCR primer pairs, preferentially in a temperature-gradient PCR machine.
  4. Mix all primers equally at 1 μM in 0.5X TE buffer.
  5. Keep at -20°C for long term storage.
  6. Multiplex PCR 5X Master Mix is stable for 15 freeze-thaw cycles when stored at -20°C.
  7. Multiplex PCR 5X Master Mix is stable at 4°C for three months. So, for frequent use, an aliquot may be kept at 4°C.

References

  1. Beggs et al. (1990). Hum. Genet.. 86, 45-48.
  2. Krenke et al. (2002). J. Forensic Sci.. 47, 773-785.
  3. Sun, Y. et al. (1993). Biotechniques. 15, 372-374.

Protocols, Manuals & Usage

Protocols

  1. Multiplex PCR Guidelines for Multiplex PCR 5X Master Mix
  2. Quick Protocol for Multiplex PCR 5X Master Mix

Usage & Guidelines

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What ends will my PCR products have?
  2. How should I determine the appropriate annealing temperature for my reaction?
  3. What should the final primer concentration be in my PCR?
  4. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
  5. What are the stability and storage requirements for NEB's Taq and OneTaq DNA Polymerases?
  6. Can Taq/OneTaq® DNA Polymerases be used for nick translation?
  7. How can I optimize my PCR when using Taq DNA Polymerase?

Troubleshooting

Tech Tips

Test each primer set individually before combining into a multiplex reaction. The concentration of some primer pairs may need to be adjusted to achieve consistent and robust amplification across all amplicons in the mix.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Trademarks

NEW ENGLAND BIOLABS® and THERMOPOL® are registered trademarks of New England Biolabs, Inc.
IGEPAL® is a registered trademark of Rhodia Operations.
TWEEN® is a registered trademark of Uniqema Americas, LLC.

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