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  • Multiplex PCR 5X Master Mix


    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: 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 2-Log DNA Ladder (NEB #N3200).
    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: 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.

    Advantages and Features


    • Multiplex with Taq

    Properties and Usage

    Storage Temperature


    Buffer 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
    100 units/ml Taq DNA Polymerase
    pH 8.9@25°C

    Heat Inactivation


    Unit Assay Conditions

    1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 200 µg/ml activated Calf Thymus 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.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • PCR Amplification (Multi-Plex PCR, Master Mix) :
      The polymerase master mix is tested in a multi-plex polymerase chain reaction (PCR) using a control template and specific primer sets, resulting in the expected 15 products.


    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.


    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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is the stability of Multiplex PCR 5X Master Mix?
    2. How should I set up a PCR using Multiplex PCR 5X Master Mix?
    3. What type of DNA ends result from a primer extension reaction or a PCR using Taq DNA Polymerase?
    4. Does the presence of Ca2+ inhibit PCR?
    1. Multiplex PCR Guidelines for Multiplex PCR 5X Master Mix
    2. Quick Protocol for Multiplex PCR 5X Master Mix

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    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.