Multiplex PCR NZYProof 2x Green Master Mix (MB337)



Description: Multiplex PCR NZYProof 2× Green Master Mix is a premixed ready-to-use solution designed for the simultaneous amplification of multiple DNA fragments (up to 15 targets) in a single tube with high accuracy. The Master Mix contains an engineered proofreading DNA polymerase displaying a hot-start-like PCR capacity and high processivity (fast polymerisation), while reducing errors due to its 3´→5´ exonuclease activity. The enzyme is inactive at room temperature, avoiding extension of non-specifically annealed primers or primer-dimers, and thus ensuring highly specific and sensitive multiplex PCR amplification. In addition, the master mix contains dNTPs and reaction buffer at optimal concentrations for efficient multiplexing of targets ranging in size from 70 bp to 2.5 kb over a broad range of primer and template concentrations. MgCl2 final concentration is 1.5 mM, allowing the implementation of a comprehensive variety of PCR protocols. Reactions assembled with Multiplex PCR NZYProof 2× Green Master Mix may be directly loaded onto agarose gels, which simplifies the workflow. There are two dyes (blue and yellow) in the mix that allow monitoring the progress of electrophoresis.

– High performance in multiplexing up to 15 primer pairs
– Direct Gel Loading
– Proofreading activity
– High sensitivity and specificity
– Minimal optimization requirements
– Leaves an A-overhang

– Multiplex PCR
– High-Fidelity PCR
– Typing/ Detection
– Generation of products for TA cloning


Product length:0-3 kb
Hot-start-like capacity:yes
Extension time:30 sec/kb at 72 °C
3´→5´ activity:yes
Product overhang:blunt
Storage conditions:Store at -20 ºC
Shipping conditions:Shipped at 4 ºC to dry ice

Multiplex PCR NZYProof 2× Green Master Mix (0.2 U/μL)

Product Brochure EN
Certificate of Analysis 17051

1. What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature/ primer concentration. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (3 ° to 5 °C lower than Tm). Optimise annealing temperature using temperature gradient PCR. Increase primer concentration for lower yield amplicons.
b) Inadequate cycling conditions. Increase extension time and/or number of cycles.
c) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
d) Inadequate amount of template DNA. Titrate template amount. We recommend starting with 20-50 ng of genomic DNA.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.25 mM increments. (Note: MgCl2 is not provided in separate tubes).

2. How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another pool of primers, avoiding complementary sequences. Ensure that each primer pair originates a single product without primer-dimers in individual PCR reactions. If necessary, reduce primer concentration. Reduce the number of cycles.

3. What is the maximum size of amplicon using Multiplex PCR NZYProof 2x Green Master Mix?
Multiplex PCR NZYProof 2x Green Master Mix allows efficient multiplexing of targets ranging in size from 70 bp to 2.5 k.

4. Which type of DNA ends are generated in the PCR products using Multiplex PCR NZYProof 2x Green Master Mix?
Resulting PCR products have blunt-ends and are suitable for cloning with NZYTech´s NZY-blunt PCR cloning kit (MB121).

5. I don’t distinguish separate bands using agarose gel electrophoresis. What should I do?
When separating samples through agarose gel electrophoresis, please take into account that both concentration and grade of agarose are important when working with closely-sized fragments. NZYTech offers a vast portfolio of agaroses with different specifications to cover a wide range of needs. Please visit to identify the agarose that most suits your experiment.

6. How should I store my Multiplex PCR NZYProof 2x Green Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.

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