2 × SYBR Green qPCR Master Mix (High ROX) 1mL
- Stock: In Stock
- Model: 0470
Product Description/Introduction
2×SYBR Green qPCR Master Mix (High ROX) is a ready-to-use solution optimized for quantitative real-time PCR and two-step real-time RT-PCR. The master mix includes Hot Start Taq DNA polymerase and dNTPs in an optimized PCR buffer to ensures PCR specificity and sensitivity. The SYBR Green I intercalating dye allows for DNA detection and analysis without using sequence-specific probes. Only template and primers need to be added. The use of 2×SYBR Green qPCR Master Mix (High ROX) in real time PCR ensures reproducible, sensitive and specific quantification of genomic, plasmid, viral and cDNA templates.
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. Recommended PCR reaction systems
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 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. 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 | 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. 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 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
Brand of PCR machine | (None ROX) | (Low ROX) | (High ROX) |
ABI Thermo life | PikoRealTM Cycler | 7500/7500 Fast， ViiA 7™ QuantStudio™ series | 5700/7000/7300/7700/7900/ 7900HT/7900 HT Fast，StepOne™，StepOne Plus™ |
Stratagene | Mx3000P®/3005P™/4000™ | ||
Bio-Rad | All series | ||
Eppendorf | Realplex 2s，Mastercycler®ep realplex | ||
IIIumina | Eco QPCR | ||
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