Super Green qPCR mix (~Sybr Green  qPCR mix)

(None/Low/High ROX & Universal)

Product Description

 Super Green qPCR Master Mix is a ready-to-use cocktail containing all components except primers and template. The 2X master mix contains Taq DNA polymerase, dNTPs, MgCl₂, SYBR Green I, Rox or No Rox and stablizers. In the formulation, for HotStart, Taq DNA Polymerase is chemically modified and its activity is completely blocked until the first denaturation step in PCR program. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency. For easy and avoiding potential error manipulation, the products are provided in three formats: no Rox, low Rox, high Rox. Customers could choose suitable product format based on their qPCR instruments used.

Product Components:

F021-NOROX contains no ROX, F021-LOWROX contains low ROX，F021-HIGHROX contains high ROX.

Following table is helpful for choosing right product formats

Storage Condition:

Store at -20℃avoiding light, the master mix should be not stored at 4℃ for longer time after being thawed. The mix can tolerate 30 cycles freeze and thaw without affecting Ct value for sample analysis, keep at -20℃ if no plan to use in certain time. Please blending the master mix before aliquot.

Experimental Procedure

1. Set up reaction in qPCR tube as follow:

Quantity of components in the reaction could be adjusted as needed to achieve better results:

 1)  Generally 0.2 μM primer concentration is suitable, but when results are not satisfied,  trying primer concentration between 0.1-1.0 μM range

2) qPCR method is very sensitive, accuracy of added temple is essential, recommending using diluted templates to reduce to increase aliquot accuracy, and the result is reproducible.

3) If templates are undiluted cDNA from standard reverse transcription reaction, the volume of template is no more than 10% of the reaction volume.

2. Running qPCR reaction as following

2.1Pre-denature condition is suitable for most of reactions, if templates are complicated, extend to 10 min.

2.2for less 300 bp fragment amplification, 30 second extending time is enough, for large than 300 bp fragment amplification,  60 second extending time is recommended.

2.3Melting curve collecting program depends on instrument’s model, please choose acquiescence for the model

Quality Control

Purity detection：all components are analyzed without exo- and endo-nulease and nuclear acid.

Optimizing reaction

Best reaction condition should have following characteristic: single melting curve, amplification efficiency is almost 100%, lower Ct value (high amplification efficiency), if reaction is not as expected under acquiescence condition, reaction condition could be optimized as following ways.

1. Primer concentration and reaction: when primer concentration is between 0.1～1.0 μM, higher primer concentration leads non-specific amplification, but amplification efficiency is increased.

2. Amplification program and reaction: To increase amplification specificity, increase annealing temperature and extending amplification time

3. To increase amplification efficacy, change two step amplification to three step and increase extending time.

NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.