2 × SYBR Green qPCR Master Mix (None ROX) 1mL
- Stock: In Stock
- Model: 0472
Product Description/Introduction
2×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 quantification area, accurate quantification of target genes, good reproducibility and high confidence.
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 apparatus;
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×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.
2. PCR reaction program (can be adjusted appropriately according to the instrument)
A. Two-step process * | B. Three-step process* | ||||||||
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 | 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. Reagents are mixed well before use.
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 tips 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")
