QIAGEN Multiplex PCR Kit

For highly specific and sensitive multiplex PCR without optimization requirements

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QIAGEN Multiplex PCR Kit (100)

Cat. No. / ID: 206143

For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl₂, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml)

Product

QIAGEN Multiplex PCR Kit (100)

QIAGEN Multiplex PCR Kit (1000)

El QIAGEN Multiplex PCR Kit está concebido para su uso en aplicaciones de biología molecular. Este producto no está concebido para el diagnóstico, la prevención ni el tratamiento de enfermedades.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

No optimization required
High specificity and sensitivity with a built-in hot start
Highly suited for many types of multiplex PCR applications
Easy to use and cost-effective

Product Details

The QIAGEN Multiplex PCR Kit is available in a convenient ready-to-use master mix format. The QIAGEN Multiplex PCR Master Mix includes HotStarTaq DNA Polymerase and a unique PCR buffer containing the novel synthetic Factor MP. Together with optimized salt concentrations, this additive stabilizes specifically bound primers and enables efficient extension of all primers in the reaction without the need for optimization. Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is also supplied.

Performance

The QIAGEN Multiplex PCR Kit outperformed kits tested from other suppliers and ensures highly specific and sensitive multiplex PCR amplification (see figure " Successful 16-plex PCR "). The kit can be successfully used for various multiplex applications such as typing of transgenic organisms (see figure " Genotyping transgenic mice") and microsatellite analysis (see figure figure " Successful microsatellite analysis "). The master mix includes HotStarTaq DNA Polymerase for efficient amplification of multiple targets in parallel. Amplfication effiency is further improved by an innovative PCR buffer, also included in the master mix. The unique buffer ensures PCR specificity over a wide range of PCR conditions, minimizing the need for optimization. Suboptimal PCR can be improved with Q-Solution — an additive for the amplification of GC-rich templates — also provided with the kit .

HotStarTaq DNA Polymerase specifications

Concentration: 5 units/µl
Recombinant enzyme: Yes
Substrate analogs: dNTP, ddNTP, dUTP, biotin-11-dUTP, DIG-11-dUTP, fluorescent-dNTP/ddNTP
Extension rate: 2–4 kb/min at 72°C
Half-life: 10 min at 97°C ; 60 min at 94°C
Amplification efficiency: ≥10⁵ fold
5'–>3' exonuclease activity: Yes
Extra A addition: Yes
3'–>5' exonuclease activity: No
Contaminating nucleases: No
Contaminating RNases: No
Contaminating proteases: No
Self-priming activity: No >

See figures

Successful 16-plex PCR.

Genotyping transgenic mice.

Successful microsatellite analysis.

Principle

The QIAGEN Multiplex PCR Kit is the first kit specifically developed for multiplex PCR and is provided in an easy-to-use master-mix format. QIAGEN Multiplex PCR Master Mix contains preoptimized concentrations of HotStarTaq DNA Polymerase and MgCl^₂, plus dNTPs and an innovative PCR buffer specially developed for multiplex PCR. The kit enables success in multiplex PCR at the first attempt. There is no need to optimize reaction conditions (e.g., the concentrations of primers, Mg²⁺, and Taq DNA polymerase) and cycling parameters due to unique preoptimized reagents included in the kit.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase is a modified form of Taq DNA polymerase and has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle. HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program.

Multiplex PCR Buffer

This special buffer contains an optimized combination of K⁺ and NH₄⁺, as well as the unique PCR additive, Factor MP, which increases the local concentration of primers at the template. Together with K⁺ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase (see figure " Stable and efficient primer annealing"). The innovative buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and (NH₄)₂SO₄, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg²⁺ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg²⁺ concentration is therefore often minimal or not required.

Q-Solution

Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA, is also provided with HotStarTaq DNA Polymerase. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates. Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed.

See figures

Stable and efficient primer annealing.

Procedure

The QIAGEN Multiplex PCR Kit is provided in a ready-to-use, preoptimized master mix for greater convenience. Use of a master mix saves time, simplifies handling for reaction setup, and increases reproducibility by eliminating many possible sources of pipetting errors and contamination — pipetting steps are minimized and tedious calculations are eliminated. Only primers and template need to be added to prepare the final amplification mix. The master mix can be stored at
2–8°C, allowing even faster setup of multiplex PCR assays. The streamlined, step-by-step protocol provided with the kit ensures fast and easy PCR setup. Reactions can be set up at room temperature, ensuring greater convenience and ease of use. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs.

Applications

The QIAGEN Multiplex PCR Kit is highly suited for various multiplex applications, including:

Typing and analysis of transgenic organisms
Amplification and analysis of microsatellites
Typing and detection of bacteria and viruses
Amplification of multiple DNA regions for SNP analysis

Supporting data and figures

Successful 16-plex PCR.

Multiplex PCR of 16 targets (99–955 bp) was carried out for 35 cycles using standard conditions for the QIAGEN Multiplex PCR Kit, without further optimization or using a variety of conditions with a hot-start DNA polymerase from Supplier A_II. [A] Comparison using 2.5 units per 50 µl reaction of the hot-start DNA polymerase from Supplier A_II and with the indicated Mg²⁺ concentrations. [B] Comparison using the optimized Mg²⁺ concentration (3.5 mM) for the hot-start DNA polymerase from Supplier A_II (part A) and the indicated amounts of enzyme per 50 µl reaction. Successful results were ensured with the QIAGEN Multiplex PCR Kit. M: markers.

Specifications

Features	Specifications
Applications	PCR, RT-PCR, multiplex PCR, typing, detection
Enzyme activity	5' -> 3' exonuclease activity
Reaction type	PCR amplification
With/without hotstart	With hotstart
Single or multiplex	Multiplex
Real-time or endpoint	Endpoint
Mastermix	Yes
Sample/target type	Genomic DNA and cDNA

Resources

Download Safety Data Sheets for QIAGEN product components.

Second edition — innovative tools

Addressing critical factors and new solutions

For fast and efficient multiplex PCR without optimization

Publications

Screening for clinically significant non-deletional alpha thalassaemia mutations by pyrosequencing.

Haywood A; Dreau H; Timbs A; Schuh A; Old J; Henderson S;

Ann Hematol; 2010; 89 (12):1215-21 2010 Jun 22 PMID:20567827

Pyrosequencing-based strategy for a successful SNP detection in two hypervariable regions: HV-I/HV-II of the human mitochondrial displacement loop.

Anjum GM; Du W; Klein R; Amara U; Huber-Lang M; Schneider EM; Wiegand P;

Electrophoresis; 2010; 31 (2):309-14 2010 Jan PMID:20084631

In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.

Zhai W; Jeong H; Cui L; Krainc D; Tjian R;

Cell; 2005; 123 (7):1241-53 2005 Dec 29 PMID:16377565

Isolation of genomic DNA from buccal swabs for forensic analysis, using fully automated silica-membrane purification technology.

Hanselle T; Otte M; Schnibbe T; Smythe E; Krieg-Schneider F;

Leg Med (Tokyo); 2003; 5 Suppl 1 :S145-9 2003 Mar PMID:12935575

FAQ

The concentration of the agarose gel for separation of multiplex PCR products should be appropriate for the overall size of products generated and can be adjusted for resolving small size differences between PCR fragments. For optimal results, we recommend the use of 1x TAE buffer for preparation and running of the gel. Use the general guidelines listed in the table below for choosing the percentage of agarose.

Minimum difference in size of PCR products	Maximum size of fragments	Concentration of agarose
>200 bp	2000 bp	1.3%
>100-200 bp	1000 bp	1.4-1.6%
>50-100 bp	750 bp	1.7-2.0%
20-50 bp	500 bp	2.5-3.0%
<20bp*	250 bp	3.0-4.0%

*Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard molecular-biology–grade agarose. For separation of fragments that differ in size by less than 20 bp, we recommend using high-resolution agarose, for example MetaPhor® agarose (FMC Bioproducts). For more information, visit www.cambrex.com.

Please refer to the QIAGEN Multiplex PCR Handbook for additional information, and for details on successful multiplex PCR using the QIAGEN Multiplex PCR Kit.

FAQ ID -800

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.

The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.

Spectrophotometric conversions for nucleic acid templates

1 A₂₆₀ unit*	Concentration (ug/ml)
Double-stranded DNA	50
Single-stranded DNA	33
Single-stranded RNA	40

*Absorbance at 260 nm = 1

Molar conversions for nucleic acid templates

Nucleic Acid	Size	pmol/ug	Molecules/ug
1 kb DNA	1000 bp	1.52	9.1 x 10¹¹
pUC 19 DNA	2686 bp	0.57	3.4 x 10¹¹
pTZ18R DNA	2870 bp	0.54	3.2 x 10¹¹
pBluescript II DNA	2961 bp	0.52	3.1 x 10¹¹
Lambda DNA	48,502 bp	0.03	1.8 x 10¹⁰
Average mRNA	1930 nt	1.67	1.0 x 10¹²
Genomic DNA
Escherichia coli	4.7 x 10⁶*	3.0 x 10^-4	1.8 x 10⁸**
Drosophila melanogaster	1.4 x 10⁸*	1.1 x 10^-5	6.6 x 10⁵**
Mus musculus (mouse)	2.7 x 10⁹*	5.7 x 10^-7	3.4 x 10⁵**
Homo sapiens (human)	3.3 x 10⁹*	4.7 x 10^-7	2.8 x 10⁵**

* Base pairs per haploid genome

** For single-copy genes

FAQ ID -74

Always start with the cycling conditions specified in the handbook for the QIAGEN Multiplex PCR Kit to guarantee best performance.

FAQ ID -287

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

FAQ ID -204

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).

FAQ ID -961

Not necessarily. In a lot of cases, the uniquely formulated PCR Buffer provided in the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and QIAGEN Multiplex PCR Kits provides optimal amplification of specific PCR products. The usefulness of Q-Solution needs to be determined empirically for each primer/template setup, by running parallel PCR reactions with and without Q-Solution under the same cycling conditions.

Q-Solution changes the melting behavior of DNA and will often improve a suboptimal PCR caused by templates that have a high degree of secondary structure or high GC-contents. For more details on the effects of Q-Solution on PCR amplification, please see the Q-Solution sections of the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Handbooks.

FAQ ID -380

The exact composition of the QIAGEN Multiplex PCR Buffer supplied in the QIAGEN Multiplex PCR Kit is proprietary. The buffer contains a specially developed balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction, thereby facilitating the amplification of multiple PCR products in a single tube.

The QIAnews article 'Highly efficient multiplex PCR using novel reaction chemistry' provides additional details and describes the advantages of this buffer in contrast to conventional PCR reagents.

FAQ ID -289

Yes, the Multiplex PCR Master Mix will freeze at -20°C. Freeze-thaw cycles up to 10 times will not negatively influence the performance of the kit. The 2x QIAGEN Multiplex PCR Master Mix can also be stored at 2-8°C for up to 6 months.

FAQ ID - 525

Yes. Please see Table 3 in our brochure Maximizing PCR and RT-PCR success. We tested the effects of different inhibitory substances in a number of PCR systems. We also analyzed the effect of including different volumes of reverse transcription (RT) reaction mixtures in PCR. Please see the table below for a list of commonly encountered template impurities and their inhibitory effects on PCR.

Impurities showing inhibitory effects on PCR

Substance	Inhibitory concentration
SDS	>0.005% (w/v)
Phenol	>0.2% (v/v)
Ethanol	>1% (v/v)
Isopropanol	>1% (v/v)
Sodium Acetate	≥5 mM
Sodium Chloride	≥25 nM
EDTA	≥0.5 mM
Hemoglobin	≥1 mg/ml
Heparin	≥0.15 i.U./ml
Urea	>20 mM
RT reaction mixture	≥15%

FAQ ID -818

Yes. Precipitates may form when the buffer freezes. We recommend that you thaw the buffer on ice, then vortex the tube at room temperature until the precipitate has re-dissolved. Do not centrifuge the tube. Do not heat the buffer.

FAQ ID -216

No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.

FAQ ID -565

The DNA yield obtained in a PCR reaction depends on the size of the amplicon, design of the primers, starting amount of template and primers, amplification efficiency, reaction volume, numbers of PCR cycles etc. Therefore it is really difficult to predict what yield to expect. Nevertheless, in our experience, approximately 1 µg is a good guess for most cases.

FAQ ID -750

To determine the optimal annealing temperature for a PCR assay, a Temperature Gradient experiment should be performed. To do this, you will set up several PCR reactions in duplicate for the same primer/template combination, using the same PCR chemistry, and subject each of the reactions to a slightly different annealing temperature within a specified range. If a thermal cycler with a temperature gradient function can be used, you can simply program a temperature range for adjacent wells in the cycling block. If no cycler with a gradient function exists in your lab, you will either have to perform duplicate reactions at different temperatures in different machines (if available), or back to back in the same machine.

FAQ ID -288