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Golden qPCR TaqMan Probe Master Mix (2x) 

Golden qPCR TaqMan Probe Master Mix(2×)is a special mixture for Taqman probe real time PCR Which includes the hot-start DNA polymerase, PCR Buffer, MgCl2, dNTPs and all other components required for quantitative real-time PCR, except DNA template, primers and probes.
Cat. No. Size Price Quantity  
TB20002A 5x1 mL USD 109
TB20002B 25x1 mL USD 382
Tel: +1 (973) 789-8318Email: [email protected]

Description

Highlights

  1. High sensitivity and specificity. The chemically modified hot-start DNA polymerase and the optimized buffer in the Mix eliminate non-specific amplification and formation of primer dimers. This ensures reproducible, sensitive and specific quantification of template.
  2. Convenience. A premixed, ready-to-use solution for Taqman probe real time PCR.
  3. Slim well-to-well variation(without ROX)

Overview

Golden qPCR TaqMan Probe Master Mix(2×)is a special mixture for Taqman probe real time PCR Which includes the hot-start DNA polymerase, PCR Buffer, MgCl2, dNTPs and all other components required for quantitative real-time PCR, except DNA template, primers and probes.

Basic Principles

The PCR reaction exploits the 5' nuclease activity of DNA Polymerase to cleave a TaqMan probe which anneals within a DNA region amplified by a specific set of primers during PCR. The TaqMan probe contains a reporter dye at the 5' end of the probe and a quencher dye at the 3' end. The quencher dye quenches the fluorescence emitted by reporter dye via FRET when the probe is intact. During the PCR, as the DNA polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the DNA polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from the reporter and breaks the quenching effect of the quencher. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Shipping and Storage

  • Shipping: blue ice
    Storage: -20°C for 12 months or 4°C for 2 months (Avoid repeated freeze-thaw cycle)

Notes

  1. FOR RESEARCH USE ONLY. NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE
  2. An initial denaturation step of 10 min at 95°C to activate Hot Start Taq DNA polymerase is necessary.
  3. ROX dye is not provided.

Instructions

1.Materials Required but not Provided

(1) Primer

Primers with good reactivity is critical to successful real-time PCR. Follow the guidelines below to design primers:

A. The length of primers should be 18-25 bp

B. The base composition should be 40-60% (G+C)

C. The Tm of forward primer and reverse primer should not differ greatly (< 2°C) and in the range of 59-68°C

D. The optimal amplification product size should be 50-250 bp.

(2) Probe

  • A. The probe should be in close proximity to the forward primer.
    B. The length of probe should be 25-35bp.
    C. Probes should have a Tm 10°C higher than the primers, usually 68-70°C.
    D. GC content of the probe should be 40%-70% and avoid a G at the 5' end.
    E. The content of C should be higher than that of G in the probe.

(3) DNA or cDNA Templates

A. DNA templates(genomes,plasmids or PCR product etc.)should be less than 10ng.

B. The pH of elution buffer should be 7.0-9.0

C. Template volume should not exceed 10% of the PCR mixture.

(4) ddH2O

  • RT-PCR grade water/Nuclease free water

(5)PCR plastic consumables

  • Always use sterile pipette tips (with filters better), tubes, strips and 96-well plates designed for real time PCR.

2.Protocol

Depending on the nature of your amplicon and instrument capability, you may choose the following protocols or follow the procedures provided in the manual of the respective instrument. The following protocols are based on ABI PRISM 7500/7300 Real time PCR System.

(1)Reaction Setup

Prepare the PCR mixture shown below. Add the template after dispensing.

Reagent 50 µl System 20 µl System Final Conc.
Golden qPCR TaqMan Probe Master Mix 25 µl 10 µl 1 ×
Forward Primer(10 µM) 1 µl 0.4 µl 0.2 µM
Reverse Primer(10 µM) 1 µl 0.4 µl 0.2 µM
Water 18-X µl 7.2 -X µl NA
Template X µl X µl NA
Total 50 µl 20 µl NA

(2) Dispensing the master mix and adding template

Vortex the master mix thoroughly and dispense the mix into real-time PCR tubes or wells of the PCR plate.Add the remaining x μl of template DNA to each reaction vessel containing the master mix and seal the tubes/plate.

(3) Start the reaction.

  • The 2-step PCR standard protocol is recommended.
2-step PCR Standard Protocol
Stage 1 Initial Denaturation 1 Cycle 95 °C 10 min*
Stage 2 Cycling Stage 40 Cycles 95 °C 15 s
60 °C 1 min
Stage 3 Dissociation Stage
  • *Initial denaturation step of 10 min is necessary to activate Hot Start Taq DNA polymerase.

(3) Melting / Dissociation Curve Analysis

  • The melting curve analysis should be performed according to the recommendations of each real time cycle.

3.Optimization

(1) Template

It is important that the DNA template is purified. Any nuclease, DNA polymerase inhibitor or DNA-binding protein in the template may interfere PCR. To those G+C rich templates, glycerol (5-10% Final conc.) and betaine (0.5-1 M) may be useful to reduce the nonspecific amplification.

(2) Primer

Final primer concentration of 0.2 µM is suitable for most reactions. However, to determine the optimal concentration we recommend titrating in the range 0.1-1 μM. Reducing the primer concentration raises reaction specificity, whereas increasing the primer concentration raises amplification efficiency. The final primer concentration can be decreased to 0.05-0.1 µM when nonspecific amplification or primer dimer occurs and the final primer concentration can be increased to 0.2-1.0 µM when the amplification efficiency is low.

(3) DNA polymerase,Mg2+ and dNTPs

The concentration of DNA polymerase,Mg2+ and dNTPs has been optimized.

(4) PCR Cycling Conditions

▴ The 2-step PCR standard protocol is recommended and perform 3-step PCR when using a primer with a low Tm value or 2-step PCR is not feasible.

▴ Optimize annealing temperature. Raising the annealing temperature may improve reaction specificity, whereas reducing the annealing temperature may raise amplification efficiency.

3-step PCR Standard Protocol
Stage 1 Initial Denaturation 1 Cycle 95 °C 10 min*
Stage 2 Cycling Stage 40 Cycles 95 °C 15 s
50~60 °C 30 s
72 °C* 30 s
Stage 3 Dissociation Stage
  • *Fluorescence signal is collected at the extension step (72°C).

Trouble Shooting

Problems Possible Causes Solutions
Amplification in negative control DNA contamination of reagents Use sterile pipette tips with filters and repeat with new reagents
PCR efficiency is <90%
Primer design Re-design primers or use validated pre-designed primers
Template is difficult to amplify Optimize PCR cycling conditions
PCR inhibitors present in a reaction mixture Make dilutions of template or re-purify template DNA.
Inappropriate dilutions of template Make dilutions of template afresh
PCR efficiency is <110%
nonspecific amplification 1.Reducing the primer concentration
2.Optimize annealing temperature
3.Re-design primers
Inappropriate dilutions of template Make dilutions of template afresh
R2 < 0.98
The point of the standard curve occurs at a later CT value than expected Omit the point and reanalyze
Inappropriate dilutions of template Make dilutions of template afresh

Datasheet Download

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