● Strong specificity: The buffer has a unique equilibrium system of K⁺ and NH⁴⁺ and an original H-Bond factor. The reaction system is upgraded and has higher amplification specificity.

● High sensitivity: Able to quantitatively detect the expression level of transcripts with low abundance.

● Good repeatability: The experimental results are stable and have good repeatability.

● Simple and fast: Only template, primer and ddH₂O need to be added in the premix to carry out real-time PCR reaction.

● ROX correction: ROX dyes packaged separately are more flexible to use and can ensure more accurate results.

● Widely applicable: It is widely applicable to experiments such as gene expression analysis and nucleic acid detection by SYBR Green method on various real-time PCR instruments such as ABI, Stratagene, Roche, Bio-Rad and Eppendorf.

The SuperReal PreMix Plus (SYBR Green) Kit can be stored at -30~-15°C for one year. It should be stored immediately upon receipt at -30~-15°C, protected from light. Thaw the 2× SuperReal PreMix Plus and 50× ROX Reference Dye and mix thoroughly before use. If the 2× SuperReal PreMix Plus and 50× ROX Reference Dye are thawed but not used, it is important to thoroughly mix them prior to re-freezing. (The layering of salts during the thawing process and subsequent crystallization during freezing will damage the enzyme and decrease product performance.) For frequent use, SuperReal PreMix Plus can be stored at 2-8°C for 3 months. Repeated freeze-thaw cycles should be avoided.

● The initial denaturation conditions should be 95°C 15 min to activate the hot-start enzymes.

● SuperReal PreMix Plus includes the SYBR Green I. Store the reagent in dark and avoid direct exposition to strong light during the preparation of PCR reaction mixtures.

● Gently mix the reagents by inverting the tubes and centrifuge briefly prior to use. DO NOT vortex and avoid producing bubble.

● The purity of primers is important for the specificity of PCR. Primers purified by PAGE or more superior methods are recommended.

● Typically, best amplification results can be obtained using a primer concentration of 0.3 µM. However, for individual determination of optimal primer concentration, a primer titration from 0.2 µM to 0.5 μM can be performed.

● In a 20 µl reaction volume, the amount of genomic DNA or cDNA template is usually less than 100 ng. The reverse transcription products, if used as template, should not comprise more than 20% of the total PCR reaction volume.

Unique buffer system ensures super specificity

The reaction buffer has a unique equilibrium system of K⁺ and NH₄⁺ and an original H-bond factor. The reaction system is upgraded, with extremely high amplification specificity.

Wide Applicability of Instruments

It has been proved by actual tests that it is widely applicable to gene expression analysis and nucleic acid detection experiments by SYBR Green method on ABI (Prism 7000/7300/7700/7900 HT/STEP One/7500/7500 FAST), STRATA-GENE (Mx3000P/Mx3005P/Mx4000), Roche, Bio-Rad and Eppendorf and other real time PCR instruments.

● Establish the Real-Time reaction system

Note: 2× SuperReal PreMix Plus and 50× ROX Reference Dye should be stored protected from light.

● Thaw 2× SuperReal PreMix Plus (if stored at -30~-15°C), 50× ROX Reference Dye, template, primers and RNase-free ddH₂O. Completely mix and equilibrate reagents to room temperature before use.

● Prepare a reaction solution according to the following table. All the steps should be operated on ice. 

● Real-Time Amplification

Typically, best results are obtained using a two-step PCR. However, if two-step PCR does not yield favorable results (e.g. non-specific amplification caused by low template concentration or reduced amplification efficiency induced by low Tm value) the three-step PCR is recommended.

Two-step PCR

Three-step PCR

● Close the tubes and mix samples gently. Briefly centrifugation can be performed to collect residual liquid from the walls of the tubes.

● Place the PCR tubes in the thermal cycler and then start the PCR cycle.

Take ABI 7500 Real Time PCR Instrument as an example. Optimization strategies to improve amplification efficiency in this instrument:

Optimization strategy to improve specificity in ABI 7500 Real Time PCR Instrument:

● No product, or product detected late in PCR, or only primer-dimers.

● Positive signal in no-template control (NTC).

● Primer-dimers and/or nonspecific amplification products.

● Poor repeatability of CT value.