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QIAGEN Multiplex PCR Kit

For highly specific and sensitive multiplex PCR without optimization requirements

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

Products

The QIAGEN Multiplex PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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QIAGEN Multiplex PCR Kit (1000)

Cat. No. / ID: 206145

For 1000 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 1 x 25 ml), 5x Q-Solution (1 x 10 ml), RNase-Free Water (1 x 20 ml)
SEK 23,210.00
Add to cart
Image
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)
SEK 2,790.00
Add to cart

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

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 MgCl2, 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

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

Specifications

FeaturesSpecifications
ApplicationsPCR, RT-PCR, multiplex PCR, typing, detection
Enzyme activity5' -> 3' exonuclease activity
Reaction typePCR amplification
With/without hotstartWith hotstart
Single or multiplexMultiplex
Real-time or endpointEndpoint
MastermixYes
Sample/target typeGenomic DNA and cDNA

Resources

Brochures & Guides (3)
Second edition — innovative tools
Addressing critical factors and new solutions
Kit Handbooks (1)
For fast and efficient multiplex PCR without optimization
Quick-Start Protocols (1)
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.

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

How can I separate PCR fragments that are small and very close in size on an agarose gel?

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
What should the starting template DNA quality and quantity be for PCR?

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
Can I use my own cycling conditions with the QIAGEN Multiplex PCR Kit?

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

 

FAQ ID -287
Does QIAGEN sell Q-Solution separately?
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 PolymeraseQIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.
FAQ ID -204
Do you have a protocol for polyacrylamide gel analysis of oligonucleotides?
Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).
FAQ ID -961
Is Q-Solution required for PCR with QIAGEN's PCR kits?

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
What is the composition of the QIAGEN Multiplex PCR Buffer?

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
Will the 2x QIAGEN Multiplex PCR Master Mix freeze at -20°C?
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
Have you tested the effect of inhibitors on PCR performance?

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
A white precipitate has formed in my 10x RT buffer. Is it still ok to use?
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
Can I shorten the activation time for the HotStarTaq DNA Polymerase?
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
How much DNA is obtained in the average PCR reaction?

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
How can one determine the optimal annealing temperature for a specific PCR assay?

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