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Golden qPCR EVA Green Master Mix (2x) (Dye-Based) 

Golden qPCR EVA Green Master Mix(2×) is an optimal 2× concentrated solution of hot-start DNA polymerase, dNTPs, EVA Green dye, ROX and all other components required for quantitative real-time PCR, except DNA template and primers.
Cat. No. Size Price Quantity  
TB20003A 5x1 mL USD 109
TB20003B 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.
  2. Convenience. A premixed, ready-to-use solution with Eva Green I dye.
  3. Compatible. The pre-added ROX dye allows for correction of well-to-well variation possibly caused by pipetting inaccuracies and fluorescence fluctuations. The mix is compatible with most instruments, including those not requiring a passive reference signal for data normalization.

Overview

Golden qPCR EVA Green Master Mix(2×) is an optimal 2× concentrated solution of hot-start DNA polymerase, dNTPs, EVA Green dye, ROX and all other components required for quantitative real-time PCR, except DNA template and primers. This is a premixed, ready-to-use solution with high sensitivity and specificity.

Basic Principles

Eva Green dye is the next generation DNA-binding dye, it absorbs blue light (λmax = 497 nm) and emits green light (λmax=520 nm) when binding to dsDNA. The increase in fluorescent signal is directly proportional to the quantity of exponentially accumulating PCR product molecules (amplicons), thus direct detection of PCR product is monitored by measuring the increase in fluorescence caused by the binding of EVA Green dye to ds DNA.

Shipping and Storage

  • Shipping: blue ice
  • Storage: -20°C for 12 months 4°C for 3 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 pre-added in the master mix.

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 be18-25 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.

E. The amplification product should cross an exon-exon border for gene expression analysis.

(2) 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.

(3) ddH2O

  • RT-PCR grade water/Nuclease free water

(4)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 the 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 EVA Green Master Mix(2×) 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 10min*
Stage 2 Cycling Stage 40 Cycles 95°C 15s
60°C 1min
Stage 3 Dissociation Stage
  • *Initial denaturation step of 10 min is necessary to activate Hot Start Taq DNA polymerase.
  • *check “ROX” in the “Passive Reference Dye” box in the software for those qPCR instruments detecting ROX.

(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, gly cerol (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 if 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

A. Amplification signal in negative control

  1. DNA contamination of reagents: use sterile pipette tips with filters and repeat with new reagents
  2. Primer dimer:
    1) Optimize the primer concentration and annealing temperature
    2) Re-design primers or use validated pre-designed primers.

B. Multiple peaks on the melting curve analysis:

Competition with nonspecific amplification or formation of primer dimer. The reaction conditions should be optimized or the primers should be redesigned.

Datasheet Download

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