2×Universal Blue Probe qPCR Master Mix

Product Information

Product Description

2×Universal Blue Probe qPCR Master Mix is a 2× premix for qPCR reactions using the hydrolysis probe method. The activity of Taq DNA Polymerase is blocked by antibody method, which effectively inhibits the formation of primer dimer and improves the amplification specificity and amplification efficiency. Together with the reaction Buffer optimized for Probe qPCR, a good amplification curve can be obtained in a wide quantitative region, and the quantification of target genes can be accurate, reproducible and highly reliable. It also contains a special ROX Passive Reference Dye and a blue visualization spiking tracer dye, which can be used on all qPCR instruments without the need to adjust ROX concentration on different instruments. All that is required is the addition of template, primer, probe and Nuclease-free water.

Storage and Shipping Conditions

Ship with wet ice; store at -20°C away from light, valid for 12 months..

Product Contents

Assay Protocol

Before starting

1. Real-Time PCR Instruments;

2. PCR tubes, PCR plates or accessories offer excellent qPCR performance;

3. PCR primers and probes (reference primer design principles);

4. Micropipettes and tips (autoclaved).

Procedures

1. Recommended PCR reaction systems

a. Usually, a good amplification effect can be obtained with the final concentration of 0.2 μM. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1.0 μM.

b. The amount of template added varies depending on the number of copies of the target gene, and the appropriate amount of template addition is studied by gradient dilution. The best addition amount of template DNA in the 20 μL reaction system was less than 100 ng. When the cDNA (RT reaction solution) of RT-PCR reaction was used as template, the addition amount should not exceed 10% of the final qPCR volume.

3. PCR reaction program (can be adjusted appropriately according to the instruments)

a: If amplification specificity needs to be improved, two-step procedure or annealing temperature can be used; To improve the amplification efficiency, a three-step procedure or extension time can be used.

Note

1. Mixed gently upside down before use. Do not swirl and shake for mixing to avoid air bubbles, mix the reagents well before use.

2. Reagents should be placed on ice when preparing reaction mixes.

3. Strong light should be avoided when preparing PCR reaction mixes due to fluorescent dye SYBR Green. 

4. New disposable tips should be used for preparation of reaction mixes to avoid cross contamination.

5. Avoid freeze-thawing cycles of the Master Mix and try to use it up within one month after thawing.

Compatible instruments

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900 HT Fast, StepOne, StepOne Plus, 7500/7500 Fast, ViiA 7, QuantStudio 系列, PikoRealTM Cycler;

Stratagene: Mx3000P®, 3005P, 4000;

Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon®, Opticon 2, Chromo4;

Eppendorf: Realplex 2s, Mastercycler® ep, realplex;

IIIumina: Eco QPCR;

Cepheid: SmartCycler®;

Qiagen Corbett: Rotor-Gene® series.

Roche: LightCycler Series;

Takara: Thermal Cycler Dice Series;

Analytikjena: qTOWER Series;

Hangzhou Bori: LineGene Series.

Taqman primer design Principles

1. Determine the probe first, then design the primer;

2. When designing primers, get as close to the probe as possible without overlapping the probe;

3. Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues;

4. The Tm value for each primer should be 58-60°C;

5. Avoid more than two G or C nucleotides in the last five nucleotides at 3' end to lower the risk of nonspecific priming.

6. It is preferable that primers do not contain their own complementary sequences or they will form hairpin secondary structures;

7. In order to avoid genome amplification, primers should preferably be designed across exons;

8. The Optimal amplicon length should be 50-150 bp to obtain the best PCR efficiency; 

9. No other non-specific products appeared on NCBI blast.

Taqman probe design Principles

1. The probe length should be 13-25 bp (13-30bp if conventional TaqMan probe is used);

2. The Tm value should be 65°C~70°C, usually 5~10 higher than the Tm value of the primer, to ensure that the probe is preferentially bound to the target gene during the annealing process;

3. For a primer, the guanine-cytosine (G+C) content should be between 40% and 70%;

4. G should be avoided at the 5' end of the probe because G at the 5' end will have a quenching effect, even if it is cut down.

5. In the whole probe, the content of C is significantly higher than that of G. A high content of G will have a quenching effect, in which case the paired other strand can be chosen as the probe. 

Taqman MGB Probe Design Principles

1.A reporter dye (e.g., FAM dye) is attached to the 5 'end of the probe;

2. There is a non-fluorescent quencher (NFQ) at the 3 'end of the probe;

3. The MGB moiety stabilizes the hybridized probe and effectively raises the melting temperature (Tm). This means MGB probes can be shorter than traditional dual-labeled probes, not less than 13 bp;

4. In principle, MGB probes can detect as little as one base mutation in the MGB (the MGB probe is not bind to the target gene and fluorescence signal do not appear).

For Research Use Only!

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