2 × Fast SYBR Green qPCR Master Mix (None ROX) 1mL
- Stock: In Stock
- Model: 0469
Product Description/Introduction
2× Fast SYBR Green qPCR Master Mix (None ROX) is a special 2× premix for qPCR reaction using SYBR Green I chimeric fluorescence method, which contains all qPCR components except primers and DNA templates, which can reduce the operation steps, shorten the time of adding samples, and reduce the chance of contamination. The core component is genetically engineered hot-start Taq DNA Polymerase, which effectively seals off DNA polymerase activity and prevents non-specific amplification at low temperatures by efficiently combining monoclonal antibody and Taq DNA Polymerase, with many advantages such as high specificity and high sensitivity, and is coupled with a reaction buffer optimized for qPCR. It is very suitable for high specificity and high sensitivity qPCR reaction. This product is a 2× premixed reagent containing the optimal concentration of SYBR Green I for qPCR reaction, which can obtain a good standard curve in a wide quantitative region, accurate quantification of target genes, good reproducibility, high confidence, and the fastest qPCR reaction can be completed in 30 minutes.
Storage and Shipping Conditions
Ship with wet ice. Store at -20°C without light, valid for 12 months. Avoid freeze-thaw cycles. After thawing, it can be stably stored at 4℃ for one month without light.
Assay Protocol / Procedures
Before starting
1. Real Time PCR amplification instrument;
2. Special reaction tube or reaction plate for experiment;
3. PCR primers (reference primer design principles);
4. Micropipette and autoclaving-tips;
Procedures
1.Recommend the qPCR reaction system:
Component | 20 μL rxn | 50 μL rxn | Final Concentration |
2×Fast SYBR Green qPCR Master Mix (None ROX) | 10 μL | 25 μL | 1× |
Forward Primer (10 μM)a | 0.4 μL | 1 μL | 0.2 μM |
Reverse Primer (10 μM)a | 0.4 μL | 1 μL | 0.2 μM |
Templateb | Variable | Variable | as required |
Nuclease-Free Water | Add to 20 μL | Add to 50 μL |
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.
1. PCR reaction procedure (can be adjusted according to the instruments):
Stage | Step | Number of Cycles | Temperature | Time |
Stage 1 | Predegeneration | 1 | 95℃ | 30 seca |
Stage 2 | Degeneration | 40 | 95℃ | 3-10 secb |
annealing-extension | 60℃ | 10-30 secc | ||
Stage 3 | melting curve | 1 | Instrument default Settings | |
It is recommended to use the fast procedure of fluorescent quantitative PCR instrument of the corresponding brand preferentially, and it is compatible with standard amplification procedure. The fast procedure is suitable for most genes, and the standard procedure can be tried for some genes with complex secondary structure.
a: The genome template can prolong the pre-degeneration time by 2-3 min;
b: Cycle stage: the denaturation time of standard procedure is 10 sec. If the amplified fragment is less than 200 bp, the shortest option for fast procedure is 3 sec.
c: Cycle stage: standard procedure annealing/elongation time is selected for 30 sec; If the amplified fragment is less than 200 bp, the shortest procedure could be selected as 10 sec. Over 200 bp, the recommended choice is 30 sec; For fluorescence signal collection, please set the experimental procedure according to the instruction manual of the instrument.
Note
1. Mix gently upside down before use. Do not swirl and shake to avoid bubbles. Mix the reagents well before using.
2. Reagents should be placed on ice when preparing reaction solution.
3. The product contains fluorescent dye SYBR Green, so strong light should be avoided when preparing PCR reaction solution.
4. Please using new disposable head for the preparation and packaging of the reaction solution to avoid contamination between samples.
5. Avoid repeated freeze-thawing of Master Mix and try to use it within one month after thawing.
Compatible instruments
1-250x250.jpeg "2 × Fast SYBR Green qPCR Master Mix (High ROX) 1mL")
1-250x250.jpeg "2 × Fast SYBR Green qPCR Master Mix (Low ROX) 1mL")
