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For general laboratory use.
Please centrifuge briefly before opening (volume ≤2 ml).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.
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.Content:
qPCR MultiplexMaster (blue cap)
Hot Start Polymerase Ab+, dATP, dCTP, dGTP, dUTP, reaction buffer with KCl, (NH4)2SO4, MgCl2, additives and stabilizersPCR-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
assay50 μl
assayfinal 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 μlfill 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 activation95 °C 2 min 1x Denaturation 95 °C 15 sec 35-45x Annealing and
elongation60-65 °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.
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产品组分
组分内容
型号 规格 储存温度
多重荧光定量qPCR预混液
PCR340-01 2.5ml(250次x20ul) -20°C 多重荧光定量qPCR预混液
PCR340-02 12.5ml(1250次x20ul) -20°C 多重荧光定量qPCR预混液 PCR340-03 100ml(10000次x20ul) -20°C 操作手册
1 1 室温
*注意：在使用之前，应将溶液离心至管底。
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注意事项
保存建议 推荐蓝冰或冰袋运输。当您收到产品后，按照说明书建议保存各组分。 警告 本品仅供科研使用，请勿用于临床与诊断。 -
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