2×SYBR Green qPCR Master Mix (Low ROX) 1mL
- Stock: In Stock
- Model: 0471
Product Description/Introduction
This product is a special 2× premixed solution for qPCR reaction using SYBR Green I chimeric fluorescence method. It contains all qPCR components except primers and DNA templates, which can reduce the operation steps, shorten the sample addition time and decrease the probability of contamination. The core component is the genetically engineered heat-activated Taq DNA Polymerase, which effectively blocks DNA Polymerase activity and prevents nonspecific amplification at low temperatures by efficiently binding monoclonal antibodies to the Taq DNA Polymerase. The heat-activated Taq DNA Polymerase has many advantages such as high specificity and detection sensitivity. With the reaction buffer optimized for qPCR, it is very suitable for high-specificity and high-sensitivity qPCR reactions. 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 area. The quantification of target genes is accurately, reproducible and reliable.
Storage and Handling Conditions
Transport with wet ice. Store at -20°C without light, valid for 12 months. Avoid freeze/thaw cycle. After thawing, it can be stably stored at 4℃ for one month without light.
Assay Protocol / Procedures
Preparation before experiment
1. Real Time PCR amplification apparatus;
2. Special reaction tube or reaction plate for experiment;
3. PCR primers (reference primer design principles);
4. Micropipette and pipette head (autoclaving);
Procedures
1. Recommend the qPCR reaction system:
Component | 20 μL rxn | 50 μL rxn | Final Concentration |
2×SYBR Green qPCR Master Mix (Low 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 addition varies with the copy number of the target gene in the template solution, and the appropriate amount of template addition is discussed 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 total volume of PCR reaction solution.
2. PCR reaction procedure (can be adjusted according to the instruments):
A. Two-step method * | B. Three-step method * | ||||||||
Stage | Step | Cycles | Temperature | Time | Stage | Step | Cycles | Temperature | Time |
Stage 1 | Predegeneration | 1 | 95℃ | 30 sec | Stage 1 | Predegeneration | 1 | 95℃ | 30 sec |
Stage 2 | Degeneration | 40 | 95℃ | 15 sec | Stage 2 | Degeneration | 40 | 95℃ | 15 sec |
Annealing-Extension | 60℃ | 30 seca | Annealing | 55-65℃ | 10 sec | ||||
Extension | 72℃ | 30 seca | |||||||
Stage 3 | Melting curve | 1 | Instrument default Settings | Stage 3 | Melting curve | 1 | Instrument default Settings | ||
*: 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.
a: For fluorescence signal collection, please set the experimental procedure according to the instruction manual of the instrument.
Note
1. Mixed 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.
Compatibility
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")
