The 2x TaqMan qPCR master (low rox) Mix is a special mixture for Probe real time PCR(TaqMan , Molecular Beacon etc.). Which include the HotStar DNA Polymerase, PCR Buffer, MgCl2 , dNTPs, ROX and the stabilizing agent. The HotStarTaq DNA polymerase which in the mix is inactive at room temperature, avoiding extension of non-specifically annealed primers or primer dimers and providing higher specificity of DNA amplification. The functional activity of the enzyme is activated in 10 minute incubation at 95°C. The unique reaction buffer with the special Hotstar enzyme highly increased the Amplification efficiency. The fluorescence signals is stronger, the sensitivity is higher can detect the Single copy sequences. The Rox in the mix can be used for the correction of the fluorescent signal in the qPCR. The 2x TaqMan qPCR master Mix produces a optimal results in qPCR.
The kit is applied to a wide linear range and can exactly quantitate the target gene. It can be widely applied to all qPCR instruments without using ROX as calibration dye. The applicable machine as the flowing: ABI Prism 7500/7500 Fast, QuantStudio® 3 System, QuantStudio® 5 System, QuantStudio® 6 Flex System, QuantStudio® 7 Flex System, ViiA 7 system, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000
1.Before use, please upside down gently for blending, avoid foam, and short centrifuge.
2.The Master Mix contain the ROX need to avoid the hard light when use and storage.
3.Avoid repeated freezing and thawing, frequently use can be stored at2-8°C, for long term storage can be store at -20°C.
Reaction Mixture Set Up
|2x TaqMan qPCR master Mix||25 μl||1x|
|Forward Primer (10 μM)||1 μl||0.2 μM|
|Reverse Primer (10 μM)||1 μl||0.2 μM|
|Probe (10 μM)||1 μl|
|RNase-Free water||up to 50 μl|
1.Generlly the primer conc at 0.2 μM can get a good result, the user can try from 0.1μ-1.0μM.
2.The conc of the Probe should be adjust according the actuality. User can refer to the instruction of instrument and the probe.
3.Usually user can refer to 10-100ng genomic DNA or 1-10ng cDNA as the template, but as different template have different copies, user should make a Gradient dilution to confirm the optimum conc.
Recommended thermal cycling conditions
95 °C 10 min
95 °C 15 sec 30-45 cycles
60 °C 60 sec 30-45 cycles
1.The functional activity of the enzyme should to be activated in 10 minute incubation at 95°C
2.We recommend use the two step PCR, but if can not get the good result caused by the low Tm primer and other reasons, user can use the three steps PCR, and the anneal temperature we suggest 56-60°C
We also have oligo, hot star taq, proteinase K and PCR and so on! If you need more , Please contact us as you need.