Multiplex PCR MasterMix - UNG

Cat. No.: M665822

GRADE & PURITY UNG

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Overview

Product content

M665822	Component	5×1 mL	Storage
M665822A	2×Multiplex PCR MasterMix (UNG）	5×1 mL	-20°C. Avoid freeze/thaw cycle.
M665822B	ddH₂O	5×1 mL	-20°C. Avoid freeze/thaw cycle.

Product Introduction

2×Multiplex PCR MasterMix (UNG) is a PCR premix system consisting of GoldStar Taq DNA Polymerase, Mg2+, dNTPs (including dUTP), UNG enzyme and PCR stabilizer. This product eliminates the need for complicated optimization of PCR reaction conditions and allows for easy multiplexing of PCR reactions by simple mapping of conditions.

The GoldStar Taq DNA Polymerase contained in this product is a chemically modified hot-start enzyme that effectively reduces non-specific amplification due to primer mismatches at the beginning of the PCR reaction. The unique buffer system allows all primers of the multiplex PCR reaction to be extended efficiently without additional optimization. The MasterMix also includes a GC Enhancer, which helps to achieve efficient amplification of "difficult" templates (e.g., those with high GC content). False positives due to contamination of amplification products are greatly reduced by the use of the dUTP-UNG Anti-Contamination System, which effectively removes residual contamination of PCR products. ung enzyme is inactivated during the pre-denaturation step of the PCR cycle, so that it does not interfere with the formation of new PCR products containing dU bases.

Multiplex PCR MasterMix (UNG) effectively prevents residual contamination of PCR products and is suitable for contamination-proof multiplex PCR reactions such as microsatellite analysis, genotyping and SNP detection.

quality control

No exogenous nuclease activity was examined; no host residual DNA was detected by PCR method; and the amplification performance was not significantly altered by storage at 2-8°C for 3 days.

Usage

The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.

1.PCR reaction system:

Reagents	50 μl Reaction system	Final concentration
2×Multiplex PCR MasterMix (UNG)	25 μl	1×
Primer Mix，10 µM each	1 μl	0.2 μM
Template DNA	appropriate amount
ddH2O	up to 50 μl

Note: When primer design, try to minimize the difference between the Tm of each primer, and the difference should be controlled within 5℃ as much as possible. Please use the final concentration of 0.05-0.2μM as the reference for setting the range of each primer concentration. If the amplification efficiency is not high, the concentration of primers can be increased; when non-specific amplification occurs, the concentration of primers can be decreased, thus optimizing the reaction system.

2.PCR reaction conditions:

Note: 1) In general, the annealing temperature in the experiment is 5°C lower than the melting temperature Tm of the amplification primer, and when the ideal amplification efficiency cannot be obtained, the annealing temperature is appropriately lowered; when a non-specific reaction occurs, the annealing temperature is raised, thus optimizing the reaction conditions.

2）The extension time should be set according to the size of the fragment being amplified. The amplification efficiency of GoldStar Taq DNA Polymerase included in this product is 1kb/min.

3）The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is too high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.

3. Result detection: This product does not contain dyes, after the reaction is finished, take 5µl of the reaction product and add the appropriate amount of sampling buffer for electrophoresis to detect the results.

Specifications

Specifications & Purity

UNG

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Product Line

biological_products

Application Protocols

General workflow for multiplex PCR using a 2X MasterMix (literature/general guidance; adjust per assay and refer to the product’s CoA/Spec Sheet for any item-specific instructions):

1. Primer panel design and validation

• Design primers with matched Tm (±1–2 °C), minimal cross-dimerization, and distinct product sizes.
• Validate each target in singleplex to confirm efficiency and specificity; sequence amplicons as needed.

2. Reaction setup (example for 20 µL total volume)

• Thaw MasterMix on ice, mix gently, and briefly spin.
• Combine in nuclease-free tube/plate: 10 µL 2X MasterMix; primer set (final 0.1–0.3 µM each primer to start); template DNA (1–100 ng gDNA); nuclease-free water to volume.
• Include no-template control (NTC) and positive control DNA.

3. Thermal cycling

• Use 2-step cycling (e.g., 95 °C denaturation; 60–68 °C combined anneal/extend) or touchdown strategy. Start with 30–35 cycles.

4. Post-PCR analysis

• Resolve products by capillary electrophoresis or agarose gel with appropriate ladder. Quantify by fluorescence if needed.

5. Optimization

• Rebalance primer concentrations to equalize peak/band intensities.
• Adjust annealing temperature or cycle number to improve specificity and yield.

Item-specific volumes, presence of dyes, and hot-start activation steps are not specified for this item.

Biological Roles

As a laboratory reagent, this product does not possess intrinsic biological roles in vivo. Its components enable an in vitro enzymatic process:

• Thermostable DNA polymerase (general): catalyzes template-directed DNA synthesis by adding deoxynucleotides to a primed 3′-OH, following Watson–Crick base pairing. Fidelity, processivity, and hot-start behavior vary by enzyme variant (not specified for this item).
• dNTPs: substrates incorporated into nascent DNA strands.
• Mg2+: essential cofactor coordinating the polymerase active site and triphosphate substrates.
• Buffering system: maintains pH and ionic strength across thermal cycling to preserve enzyme structure and activity.

In multiplex PCR (literature): balanced kinetics across primer pairs, controlled by primer Tm, GC content, and amplicon length, determine relative amplification efficiencies. The formulation aims to broaden the operating window for simultaneous target amplification. Specific enzyme lineage/modifications are not specified for this item.

Buffer Applications

This MasterMix serves as the complete reaction buffer system for PCR, obviating the need to assemble individual buffer, Mg2+, and dNTP components.

• Intended use: PCR amplification (multiplex format). Not a general biological buffer for cell culture or electrophoresis.
• Diluent: Use nuclease-free water to bring reactions to final volume. Avoid EDTA-containing buffers which chelate Mg2+ and inhibit polymerase.
• Additive compatibility (literature/general):
  • Low percentages of DMSO (2–5%) or betaine (0.5–1.5 M) can assist GC-rich targets; optimize carefully to maintain multiplex balance.
  • Excess salt, detergents, phenol, ethanol, or heme can inhibit amplification; ensure clean template prep.
• pH range and ionic composition: Not specified for this item; refer to CoA/Spec Sheet.

Practical tip: When converting a validated singleplex assay to multiplex, keep the same total primer concentration by dividing among targets, then empirically rebalance individual primer concentrations.

Green Alternatives

Sustainability considerations for PCR master mixes (general, literature):

• Cold-chain minimization: Some formulations are room-temperature stable or lyophilized; these reduce shipping on ice and energy use for -20 °C storage. This item’s stabilizer/RT stability is not specified; refer to CoA/Spec Sheet.
• Concentrated formats (e.g., 2X/5X): Reduce packaging and transport mass per reaction.
• Lyophilized beads/pellets: Eliminate glycerol and ship ambient, lowering carbon footprint; useful for field work.
• Waste reduction: Multiplexing itself decreases plastic consumables and reagent use by consolidating multiple singleplex reactions into one.

Trade-offs:

• RT-stable or lyophilized mixes may have different performance envelopes or require reconstitution steps.
• Preservatives that extend shelf life (e.g., azide) may impose disposal considerations; check SDS for the specific product (not specified for this item).

If sustainability is a priority, consider formats with ambient shipping and lyophilized RT-stable enzymes, provided performance meets assay requirements.

Pharmaceutical Uses

This product is supplied for research use only. It is not an API, excipient, or a clinical diagnostic reagent and has no pharmacopeial monograph assignment in this context.

• Typical industrial/lab role (general): upstream molecular biology workflows supporting research, process development, strain/construct verification, and QC assays in non-clinical settings.
• Regulatory note: For any GMP, clinical, or diagnostic use, a specifically qualified reagent and appropriate validation are required; this product is not specified for such use.

No therapeutic or clinical claims are made for this item.

Physical Properties

• Composition class: Aqueous enzyme/buffer solution for PCR (mixture).
• Appearance, concentration, dye content: Not specified for this item; refer to CoA/Spec Sheet.
• pH, ionic strength, Mg2+ concentration: Not specified for this item; refer to CoA/Spec Sheet. (Typical multiplex PCR mixes are mildly buffered near neutral to slightly alkaline, literature.)
• Density, refractive index, viscosity, MP/BP: Not applicable/reported for this multi-component aqueous mixture; values are not meaningful for routine use.
• Solubility: Water-miscible (aqueous). Not intended for use in organic solvents.
• Thermal behavior (general, literature):
  • Functional over typical PCR cycling: ~95 °C denaturation, 50–72 °C anneal/extend, with enzyme activity optimized for multiplex conditions.
  • Long-term storage at subzero temperatures preserves enzyme activity; repeated freeze–thaw can degrade performance.

All quantitative specifications (pH, salt content, stabilizers, dye) are not specified for this item; consult the current CoA/Spec Sheet for lot-specific details.

Quality and Grades

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet. Enzymatic master mixes are typically released based on functional QC rather than traditional chemical purity.
• Typical QC for multiplex PCR mixes (literature/general):
  • Functional amplification of a validated multi-amplicon panel across a dynamic input range (e.g., 1–100 ng gDNA or cDNA).
  • Sensitivity and specificity assays to confirm balanced amplification without dominant targets.
  • Absence of detectable DNase/RNase (as applicable) and bioburden limits.
  • Lot-to-lot consistency in Mg2+ and dNTP concentrations and buffer composition.
• UV/LC absorbance or contaminant specs (e.g., heavy metals, UV cutoff): not pertinent to enzyme mixes and not specified for this item.
• Stabilizers/inhibitors: If present (e.g., glycerol, trehalose, detergents), they enhance thermal stability and hot-start behavior; exact details not specified for this item.
• Documentation: For application-critical work, request the CoA/Spec Sheet for lot-specific QC metrics and recommended input ranges.

Reaction and Applications

This MasterMix is intended for simultaneous amplification of multiple DNA targets in a single PCR reaction (multiplex PCR). Applications include (literature/general):

• Genotyping and STR/SSR analysis, pathogen panels, variant screening, and amplicon library preparation where multiple loci are assayed concurrently.
• Panel consolidation to reduce pipetting steps, reagent variability, and overall runtime compared with separate singleplex reactions.

Technical considerations for multiplex success (general):

• Primer design/balancing: use similar Tm (within ~1–2 °C), avoid cross-dimers, and stagger amplicon sizes to resolve products. Start with 0.1–0.3 µM each primer; adjust concentrations to equalize band intensities.
• Amplicon size: 80–500 bp is typical for robust multiplexing; shorter targets amplify more uniformly.
• Template input: normalize DNA quantity and quality; inhibitors disproportionately affect higher-order multiplex sets.
• Cycle strategy: use a 2-step or touchdown protocol to harmonize annealing; increased extension time may be required when targeting many loci.
• Additives: DMSO/betaine can aid GC-rich regions but may disrupt balance—optimize sparingly.

Item-specific features (e.g., hot-start enzyme, dye-in mix, compatibility with qPCR) are not specified for this item; refer to CoA/Spec Sheet. Use standard clean techniques to prevent carryover contamination.

Reaction Conditions

Typical multiplex PCR cycling parameters and setup (literature/general guidance; optimize per assay):

• Reaction assembly (example using a 2X mix):
  • 2X MasterMix: 10 µL
  • Primer mix: begin with 0.1–0.3 µM each primer in final reaction; balance individual concentrations to equalize targets.
  • Template DNA: 1–100 ng genomic DNA (or 1–10 ng plasmid/cDNA) per 20 µL reaction.
  • Nuclease-free water: to 20 µL total volume.
• Cycling (illustrative):
  • Initial denaturation: 95 °C for 2–3 min.
  • 30–40 cycles of: 95 °C, 15–30 s; 55–65 °C, 20–45 s (anneal/extend or use 2-step at 60–68 °C); 68–72 °C, 20–45 s (optional dedicated extension depending on enzyme and amplicon length).
  • Final extension: 68–72 °C for 3–5 min; hold at 4 °C.
• Mg2+ and additives: start with the mix as supplied; only adjust with manufacturer-provided solutions if recommended. For GC-rich panels, 2–5% DMSO may help but can disrupt multiplex balance—re-optimize primers.
• Amplicon size: 80–500 bp preferred for higher-order multiplexing; allow ~15–30 s per 500–1000 bp if longer targets are necessary.

Item-specific optimal temperatures, ramp rates, and hot-start requirements are not specified for this item; refer to CoA/Spec Sheet.

Safety and Handling

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
• General risk profile (literature/general for PCR mixes): typically low hazard aqueous buffer containing proteins, salts, and dNTPs. Some formulations may include preservatives (e.g., trace sodium azide)—avoid contact with acids and copper/lead plumbing if present. Do not assume—verify on SDS/label.
• Primary hazards: eye/skin irritation on contact; ingestion not intended; biological contamination risk (DNase/RNase introduction) impacting experimental results.
• PPE: Lab coat, gloves, safety glasses. Avoid aerosol generation. Work in a clean area to prevent nucleic acid contamination.
• Incompatibilities: avoid strong acids/bases, oxidizers, and microbial contamination. Do not mix with organic solvents.
• First aid (overview; defer to SDS):
  • Skin/eyes: rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: move to fresh air if aerosols inhaled.
  • Ingestion: rinse mouth; do not induce vomiting; seek medical advice.
• Special handling: keep on ice during use; avoid repeated freeze–thaw (aliquot upon first thaw). Use nuclease-free tips/tubes. Decontaminate surfaces with DNA-away/10% bleach to reduce carryover risk.

Always consult the product SDS for authoritative safety information.

Solvent Selection

Not a solvent; this is an aqueous PCR reagent. Selection considerations focus on compatible diluents and additives rather than alternative solvents.

• Compatible diluent: Nuclease-free water (DNase/RNase-free). Avoid tap water or buffers containing EDTA unless explicitly recommended.
• Avoid: Organic solvents (ethanol, DMSO) unless validated; even small percentages can alter polymerase fidelity and multiplex balance. If DMSO/betaine are needed for GC-rich templates, optimize empirically (literature guidance).
• Polarity/miscibility: Fully water-based; miscible with aqueous sample matrices. Salts, detergents, or carryover phenol/ethanol from extraction can inhibit PCR.
• Practical tip: If sample contains residual ethanol/isopropanol, ensure complete evaporation before adding to the mix to preserve multiplex performance.

For formulation details (buffer composition, concentration), see the CoA/Spec Sheet; not specified for this item.

Storage and Reconstitution

• Storage conditions (from Product Data): Store at -20 °C. Avoid repeated freezing and thawing.
• Shipping: Ice chest + ice pads (from Product Data).
• Stability: Long-term stability depends on formulation; specific shelf life is not specified for this item; refer to CoA/Spec Sheet.
• Handling:
  • Thaw on ice; mix by gentle inversion—do not vortex vigorously to avoid enzyme shear/foaming.
  • Keep on cold block during setup.
  • Immediately return unused mix to -20 °C.
• Aliquoting: To minimize freeze–thaw cycles, aliquot upon first thaw into nuclease-free tubes sized for typical daily use.
• Reconstitution: If supplied as a liquid 2X MasterMix, use directly without reconstitution. If provided lyophilized (not specified), reconstitute with nuclease-free water to the stated concentration per CoA/Spec Sheet.
• Freeze–thaw guidance: Limit to as few cycles as possible; multiple cycles can reduce activity and alter multiplex balance.

For precise concentration, dye content, and shelf-life claims, consult the CoA/Spec Sheet for this SKU. For research use only.

Structure and Identity

This product is a ready-to-use enzyme/buffer blend formulated for simultaneous amplification of multiple DNA targets (multiplex PCR). It is a complex mixture rather than a single defined small molecule.

• Item type: Enzymatic PCR Master Mix for multiplex DNA amplification (typical components, literature/general): thermostable DNA polymerase, optimized reaction buffer, Mg2+ salt, balanced dNTPs, stabilizers; may include tracking dyes depending on format.
• CAS/Registry: Not applicable (mixture; no single CAS assigned).
• Molecular formula / MW / SMILES / InChIKey: Not specified for this item; refer to CoA/Spec Sheet. These identifiers are not meaningful for multi-component enzyme formulations.
• Structural features (general description):
  • Contains a thermostable DNA polymerase (protein enzyme) and buffering salts in an aqueous solution suitable for PCR cycling.
  • Mg2+ present at a working concentration appropriate for DNA polymerase catalysis (exact value not specified for this item).
• Physical form (general): typically supplied as a concentrate (e.g., 2X) or ready-to-use solution; this item’s exact concentration and presence of loading dyes are not specified for this item; refer to CoA/Spec Sheet.

Note: Because this is a formulated life-science reagent, identity is established by functional performance (PCR amplification) and QC metrics rather than by single-compound structural identifiers.

Synthetic Utility

Not a synthetic chemistry building block. As a biological reagent, its utility lies in assembling DNA amplicons enzymatically rather than in chemical synthesis.

• Relevant laboratory utility (general): preparation of PCR amplicons for cloning, sequencing, and library preparation; generating barcoded targets in a multiplex fashion to reduce reaction count.

For chemical synthesis applications, refer to other catalog categories; this MasterMix is not typically used.

Target Specificity

Not applicable. This product is a general-purpose PCR MasterMix and is not targeted to a specific biomolecular antigen or sequence. Target specificity in multiplex PCR is dictated by the user-designed primer sets and assay design.