qPCR MultiplexMaster
For research use only.
Please centrifuge briefly before opening (volume ≤2 ml).
Description:
qPCR MultiplexMaster is designed for quantitative real-time analysis of DNA samples using Dual Labeled Fluorescent Probes, e.g. TaqMan®, Molecular Beacons or FRET probes. The master mix is specially optimized for setting-up multiplex assays with ≥4 target sequences in a single tube.
The system overcomes multiplex limitations of conventional qPCR probe mixes combining an above-average robustness for a multitude of known PCR inhibitors with an excellent sensitivity for amplification of lowest template amounts.
The 2× concentrated master mix contains all reagents required for qPCR (except template and primer/probe sets) including a highly processive antibody-inhibited hot-start polymerase and ultra-pure dNTPs.
The mix provides an extremely stringent automatic hot-start allowing reaction set-up and allows temporary storage at room temperature prior to PCR.
The reaction chemistry of the mix is optimized for block-based PCR instruments. The mix can also be used with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Storage at 4 °C for up to 1 week.
Shelf Life: 12 months
Form: liquid
Concentration: 2× conc.
Content:
qPCR MultiplexMaster (blue cap)
Hot Start Polymerase Ab+, dATP, dCTP, dGTP, dUTP, reaction buffer with KCl, (NH4)2SO4, MgCl2, additives and stabilizers
PCR-grade water (white cap)
Dual-labeled DNA probes:
Real-time PCR technology based on dual-labeled DNA probes provides a high sensitive and high specific PCR system with multiplexing capability. For amplification of each target sequence a set of two PCR primers and one fluorescent DNA probe that hybridizes to an internal part of the amplicon are required. The sequence of the dual-labeled DNA probe should avoid secondary structure and primer-dimer formation.
Preparation of the qPCR master mix:
The preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified. A reaction volume of 20-50 μl is recommended for most real-time instruments. Prepare 13 volumes of master mix for 12 samples or a triple-set of 4 samples. Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
| component | 20 μl assay | 50 μl assay | final conc. |
|---|---|---|---|
| qPCR MultiplexMaster | 10 μl | 25 μl | 1x |
| each primer forward (10 μM)1) | 0.6 μl | 1.5 μl | 300 nM |
| each primer reverse (10 μM)1) | 0.6 μl | 1.5 μl | 300 nM |
| each dual- labeled probe (10 μM)2) | 0.4 μl | 1 μl | 200 nM |
| template DNA | x μl | x μl | <500 ng/assay |
| PCR-grade water | fill up to 20 μl | fill up to 50 μl | - |
1) The optimal concentration of each primer may vary from 100 to 500 nM.
2) Optimal results may require a titration of DNA probe concentration between 50 and 800 nM.
Dispensing the master mix:
Vortex the master mix thoroughly to assure homogeneity and dispense the mix into real-time PCR tubes or wells of the PCR plate.
Addition of template DNA:
Add the remaining x μl of sample/template DNA to each reaction vessel containing the master mix and cap or seal the tubes/plate. Do not exceed 500 ng DNA per reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Recommended cycling conditions:
| Initial denaturation and polymerase activation | 95 °C | 2 min | 1x |
|---|---|---|---|
| Denaturation | 95 °C | 15 sec | 35-45x |
| Annealing and elongation | 60-65 °C4) | 1 min5) | 35-45x |
4) The annealing temperature depends on the melting temperature of the primers and DNA probe used.
5) The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of up to 500 bp is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the annealing temperature may be necessary for each new combination of template DNA, primer pair and DNA probe.
Related products:
- Dual-labeled DNA probes
- Custom primers
- ROX reference dye, #PCR-351
| Cat. No. | Amount | Price |
|---|---|---|
| PCR-340S-30 % | 2 × 1,25 ml (250 reactions × 20 μl) | 125,30 € |
| PCR-340L-30 % | 10 × 1,25 ml (1.250 reactions × 20 μl) | 501,27 € |
| PCR-340-100ML | 100 ml (10.000 reactions x 20 μl) | Ask for Quotation |
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Scientific citations for Jena Bioscience pcr-340
1 scientific citations and 0 citation images found by Bioz.
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An improved duplex real-time PCR method for the systematic detection of commercially relevant crustaceans in food
Food Control, 2023-04-01 · DOI:10.1016/j.foodcont.2022.109517
Abstract: Crustaceans are known food allergens that can cause severe reactions for allergic patients.. Hitherto, methods to detect commercial relevant crustacean species often consist of many PCR systems.. In this study, we developed a highly specific duplex PCR (polymerase chain reaction) method for the detection of most commercially relevant crustaceans in food.
