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Q1 – Are all Master Mixes formulated for the same performance?
No. TaqDog 2x Green Master Mix contains additives for improved performance in difficult PCR conditions. These conditions include: templates/primers with high GC content, dirty DNA preps, low levels of PCR inhibitors, etc. For many sets of primers/templates the optimal conditions will be similar to master mixes from different companies. However, for certain primer/template sets (oftentimes ones with high GC content) the optimal conditions under each master mix diverge and the conditions for one may not work well for the other. If this is the case, an independent optimization must be made for TaqDog 2x Green Master Mix.

Q2 – Why doesn’t TaqDog 2x Green Master Mix work when I use my existing PCR protocol?
There are two likely reasons.

  1. i) In certain circumstances, optimization of the annealing temperature may be required. In the case of no visible amplification product, it is often sufficient to simply reduce the annealing temperature by 3 degrees. If still no band is visible after adjusting by 7oC, the optimal annealing temperature for the primers should be determined by running the PCR using annealing temperatures that span from the calculated melting temperature of the primers to 7oC below this temperature. If primers have different melting temperatures use the lowerof the two melting temperatures as reference.
  2. ii) If you were using a hot start master mix, such as Platinum Taq, then these types of hot start technologies require a long incubation at 95o On the other hand, the TaqDog Hot Start, as well as the TaqDog Premium 2x Green Master Mixes do not require any initial denaturation step. Simply set the denaturation temperature to 94oC for the number of cycles required for to amplify the product.

Q3 – Do I need to optimize annealing temperature when using TaqDog 2x Green Master Mix?
For new protocol development, always first optimize conditions by running at several annealing temperatures. The temperatures tested should range from the melting temperature of the primer all the way down to 7oC below the melting temperature. If primers have different melting temperatures use the lower of the two melting temperatures as reference.

Q4 – Do I need to optimize the number of cycles for PCR when using TaqDog 2x Green Master Mixes?
Yes. You should always start at an expected/anticipated number of cycles. TaqDog 2x Green Master Mixes are effective between 25 and 40 cycles.

Q5 – Are TaqDog 2x Green Master Mixes stable at room temperature?
In general, short incubations (less than 6 hrs) at room temperature will have no effect on the PCR efficiency of these master mixes. Longer incubations will results in decreased efficiency; with an expected 50% reduction after one week at room temperature.