Applied Biosystems SYBR Green mastermixes are designed to be used in real time PCR quantitation reactions where the target is amplified using a flanking primer pair set. The master mixes contain all required molecules for the reaction including buffer, dNTP, DNA polymerase and SYBR Green.
There are several improvements made over the years to enhance specificity, sensitivity, reproducibility and flexibility of SYBR green mastermixes. Please see below a comparison of application and performance of our two most popular mastermixes both offering superior sensitivity and specificity.
The newest Power Track SYBR Green Master Mix uses a two-colour tracking dye system, that enables users to monitor pipetting errors. The 2xMastermix uses a blue tracking dye while the optional sample buffer uses a yellow dye. This helps during plate setup as well making easier to follow the already loaded wells in the plate. Upon appropriate mixing of the master mix and sample, the reaction mixture will change to a green colour. Extensive testing was performed to verify that the tracking dye system doesn’t interfere with the master mixes’ performance
The Power Track SYBR Green Master Mix also offers exceptional performance compared to competitor master mixes on the market. According to the Information for Publication of Quantitative Real-Time PCR Experiments Guidelines (MIQE) it is essential to confirm specific amplification using SYBR dye based quantitation approach. Researches are required to perform Melt Curve analysis at the end of the amplification reaction to confirm that only one specific product was amplified. If there is non-specific product amplification observed researchers need to optimise reaction conditions ie. primer concentration, annealing temperature etc. to eliminate non-specific products, and if that doesn’t yield the required result there is a need for re-designing more specific primer sequences. This can add to the time required to fully optimise experiment setup before even starting with the scientific experiment. The broad Tm and primer concentration compatibility of the Power Track SYBR Green Master Mix reduces the need for several reaction optimisation steps. For optimal results, primer concentrations is recommended to be in the range of 300–800 nM.
The Power Track SYBR Green Master Mix shows high reproducibility with tight Ct values over a wide dynamic range. Tighter reproducibility allows for greater statistical significance when analysing low-abundance transcripts and smaller fold changes. For best performance we recommend to use SuperScript™ IV VILO™ Master Mix for exceptional sensitivity reverse transcribing low-abundance targets.
Performance of Power Track SYBR Green Master Mix using ten-fold dilutions of UHR cDNA ranging from 100ng to 100fg. 10µL reactions were run in quadruplicate using 60°C annealing primer Tm on Applied Biosystems QuantStudio5 Real time PCR instrument with 384 well block. PGK1 – High Expressor, ARL1, IGF2 and TCF25 - Medium Expressor
Applied Biosystems master mixes contain an inert reference dye, ROX. Using ROX or other passive reference dye in Real-time PCR is optional, however It offers and effective method in normalizing fluorescence across all samples. It removes fluorescence variations, such as those caused by bad pipetting or bubbles, from the data. In addition to ROX, PowerTrack SYBR Green Master Mix also contains UNG and dUTP to prevent contamination from previously amplified products.
PowerTrack SYBR Green Master Mix can be used in either standard or fast cycling mode and is compatible with all Applied Biosystems real-time PCR instruments. It is also compatible with the Bio-Rad CFX96, CFX384, and iQ5; Roche LightCycler 480; and Agilent Mx3005P systems.
To optimise for the best primer design and reaction conditions using Power Track SYBRS Green master mix an easy to follow experimental guideline available from the this link.
When choosing new SYBR master mixes for Real Time PCR applications we recommend to follow our Protocol to compare mastermixes. Don’t be fooled by a lower Ct offered by mastermixes, the performance is determined by assessing reaction efficiency, linearity and reproducibility when performing test on technical replicates of a dilution series.
Please don’t hesitate to contact us if you require free-sample to try our newest master mixes.
Gyongyi Lukacs PhD
Field Application Specialist