PCR Master Mix

What is a PCR or RT-PCR Master Mix?

A PCR master mix, sometimes known as super mix or ready mix, is a batch mixture of PCR reagents at optimal concentrations that can be prepared and divided among many PCR tubes or 96-well PCR plates. The master mix usually includes DNA polymerase, dNTPs, MgCl2 and buffer. Using a master mix reduces pipetting and risk of contamination, is convenient, saves time and preempts possible errors in mixing, making it ideal for high-throughput applications.

The master mix enables researchers to set up controls and test different concentrations of their target DNA or RNA templates without having to individually add precise amounts of enzymes, buffers, cofactor (usually MgCl2), water and dNTP to each reaction tube or plate well. Instead, a large master mix containing all or most of the PCR reagents is prepared once. The appropriate amount of master mix can be pipetted into tubes or plate wells and combined with any components that vary among the reactions, such as DNA or RNA templates or primers.

Large-scale PCR reactions can contain 2,000 or more individual 25 μL to 50 μL reactions. Even for a batch of 20 reactions, researchers can save time, reduce pipetting errors, and improve consistency with a master mix. These mixes circumvent reagents being entirely left out of a reaction, delivering false negatives as a result.

Our PCR Master Mix Calculator can help you plan out a master mix recipe for a custom batch of PCR reactions.

PCR Master Mix Applications

PCR master mixes are is commonly used in the following applications:

  • High-throughput PCR
  • Routine PCR

Master mixes with hot start DNA polymerase are ideal for the following applications:

  • Multiplex PCR
  • Reduction of primer dimers
  • Convenient ambient-temperature setup compatible with automation

Labs in preliminary investigation phases may vary the DNA or RNA templates while relying on the convenience of other premixed components.

PCR Master Mix Components

  • Enzyme
  • Buffer(s)
  • Cofactor - Magnesium chloride (MgCl2), is the most common. Sometimes MgSO4 is used with particular enzymes.
  • dNTP
  • Primers
  • Template DNA (if all samples will be uniform)
  • Nuclease-free or PCR-grade water

Types of Master Mixes

Many premade PCR master mixes are available to help save time and avoid errors in measurement and proportioning.

Most commercial PCR master mixes are designated for a particular type of PCR such as hot start or compatible with only certain thermocyclers. This is especially true for real-time PCR (also known as quantitative PCR or qPCR), which relies on a thermocycler that can detect the progression of the reaction based on the corresponding development of fluorescence from dye included in the master mix.

Commercial master mixes still need some preparation before use, if only to add template DNA or RNA. They may additionally require thawing, chilling, dilution with PCR-grade water, and mixing via pipette or centrifuge. Make sure to select the correct master mix to suit your experimental needs and PCR setup.

We offer many master mixes for PCR and real-time PCR that are validated for various instruments. Our handy PCR Selection Guide can help you identify an appropriate master mix or the components to make your own mix. We’ve also profiled several of our best-selling PCR master mixes by PCR style:

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Common PCR Master Mix Recipe

Basic recipe for a PCR master mix.

Volume Component Final concentration
5 µL 10X PCR Buffer 1X
1 µL dNTPs (10 mM) 200 µM
1 - 2 µL Forward primer 50 - 100 pmol
1 - 2 µL Reverse primer 50 - 100 pmol
0.5 - 1 µL
Taq DNA polymerase (5U/µL) 2.5 - 5 U
1 - 5 µL
Template DNA
1 - 200 ng
1 µL
MgCl2 (50 mM)
1 mM
Variable
Water
Add to q.s. to 50 µL
50 µL
Total volume
 

Preparing a Commercial Master Mix

Note that this typical recipe varies from mix to mix and among specific uses. Please review the provided product literature for the specific master mix or ready mix prepartion.

Volume Component Final concentration
  25 µL     2X Master Mix     1X  
  1 µL     Forward primer     50 pmol  
  1 µL     Reverse primer     50 pmol  
  1 - 5 µL     Template DNA     1 - 200 ng  
  17 - 22 µL     Water     Add to q.s. to 50 µL  
  50 µL     Total volume        

 

Standard PCR Master Mixes

ReadyMix™ Taq PCR Reaction Mix with MgCl2

ReadyMix™ Taq PCR Reaction Mix is a prepared solution containing everything needed for a PCR reaction except the specific primers and template. The mix includes our high-quality Taq DNA Polymerase, 99% pure deoxynucleotides and buffer in a 2× optimized reaction concentrate.

Roche PCR Master

The PCR Master from Roche contains all the reagents required to perform a standard PCR. All that must be added is the template DNA, the primers, and the required amount of water.

REDTaq® ReadyMix™ PCR Reaction Mix

This mix combines the performance and benefits of our REDTaq® DNA polymerase with the convenience of a ReadyMix™ PCR reaction mix. REDTaq® ReadyMix™ is a ready-to-use mixture of Taq DNA polymerase, 99% pure deoxynucleotides, reaction buffer, and an inert red dye in a 2X concentrate.

High-Fidelity PCR Master Mixes

Roche High-fidelity PCR Master

Choose the convenient High-fidelity PCR Master, a double-concentrated, ready-to-use mix that combines the Expand High Fidelity PCR system with PCR-grade dNTPs, MgCl2, and an optimized reaction buffer.

Millipore KOD Hot Start Master Mix

This is a ready-to-use 2X mixture, containing KOD Hot Start DNA polymerase, two monoclonal antibodies, ultrapure deoxynucleotides, and reaction buffer with MgSO4. This mix is optimized for convenient high-fidelity PCR.

RT-PCR Master Mixes

Roche Transcriptor High Fidelity cDNA Synthesis Kit is designed to reverse transcribe RNA with increased fidelity compared to other reverse transcriptases. The kit features Transcriptor High Fidelity Reverse Transcriptase, a blend of a recombinant reverse transcriptase and a proofreading mediating enzyme optimized for two-step RT-PCR.

SYBR® Green Quantitative RT-qPCR Kit provides a highly sensitive method for the quantitative analysis of gene expression. Our QRT-PCR ReadyMix™ combines the advantages of M-MLV RT and JumpStart Taq in a convenient ready mix.

KiCqStart® One-Step Probe RT-qPCR ReadyMix™ reagent is a ready-to-use, highly sensitive master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan® 5’-hydrolysis probes on real-time PCR systems (refer to instrument compatibility section to select the appropriate reagent for your machine).

MystiCq® microRNA cDNA Synthesis Mix contains all the components necessary to convert mature microRNAs into cDNA templates for qPCR. This kit has separate polyadenylation and reverse transcription steps that enables the possibility of optimization of the protocol, if necessary.

Real Time qPCR Master Mixes

Probe-based qPCR

The FastStart TaqMan® Probe Master from Roche is a ready-to-use, 2× concentrated master mix that contains all the reagents (except primers, probe, and template) needed for running quantitative, real-time DNA-detection assays, including qPCR and two-step qRT-PCR, in the hydrolysis probe detection format.

Roche EagleTaq Universal Master Mix (ROX) is a ready-to-use, 2× concentrated PCR master mix that contains all the reagents (except primers, probes, and template) needed for performing quantitative, real-time PCR hydrolysis probe reactions. It contains a special ROX reference dye which makes it suitable for all real-time instruments on which a ROX reference dye is needed for quantitative analysis.

SYBR® Green PCR

SYBR® green master mixes are suitable for real-time PCR. They employ an asymmetrical cyanine dye. Our most popular SYBR® green mix is Roche’s FastStart SYBR® Green. The FastStart SYBR® Green Master is a ready-to-use reagent mix that simplifies the preparation of reactions for qPCR and two-step qRT-PCR using the intercalating dye SYBR® Green I for DNA detection and analysis.

KiCqStart® qPCR ReadyMix™ Reagents

KiCqStart® qPCR ReadyMix™ master mixes are provided ready to use in a concentrated format. They include buffer, stabilizers, and hot-start Taq. Use them in fast or conventional cycling protocols on any thermal cycler. Choose from SYBR® green or probe-based mixes.

The KiCqStart® SYBR® Green qPCR ReadyMix™ master mix is compatible with Bio-Rad, Cepheid, Eppendorf, Illumina, Corbett, and Roche systems. Components include a 2× reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, dTTP), KiCqStart® Taq DNA Polymerase, SYBR® Green dye, and stabilizers.

Low ROX™ works with Low ROX™ for ABI and Stratagene instruments.

LuminoCt™ qPCR Reagents

Based on a highly optimized ReadyMix™ chemistry, LuminoCt™ qPCR reagents deliver unsurpassed assay speeds without sacrificing accuracy, precision, or sensitivity. And best of all, LuminoCt™ reagents work seamlessly with most qPCR instruments, so optimization of reaction conditions is kept to an absolute minimum. Often, users can simply choose their kit, build their assay, and begin. Generating the best possible qPCR data has never been faster or easier.

The LuminoCt™ SYBR® Green qPCR ReadyMix™ reagent is designed to deliver unsurpassed assay speeds without sacrificing accuracy, precision, or sensitivity. Extensive design and validation of the ReadyMix™ chemistry has virtually eliminated the need for optimization when LuminoCt™ reagent is used in conjunction with properly designed primers. Inclusion of JumpStart™ Taq antibody in the ReadyMix™ reagent eliminates polymerase activity at ambient temperatures without causing the performance issues associated with chemically modified hot-start Taq.

The LuminoCt™ qPCR ReadyMix™ kit is designed to perform probe-based qPCR assays on commonly available real-time instrument platforms. The ReadyMix™ reagent requires the addition of reference dye (provided in kit) when being used on real-time instruments that require ROX passive reference dye for normalization of qPCR assays.

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