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Q1 – Are all Master Mixes formulated for the same performance?
No. TaqDog 2x Green Master Mix contains no special additives so that performance for basic PCR using standard primer sets should be straightforward with few, if any changes to your existing protocols. However, every manufacturer’s buffers/polymerase composition can behave differently, especially under more challenging PCR conditions. These conditions include: templates/primers with high GC content, dirty DNA preps, low levels of PCR inhibitors, etc. For these challenging conditions the optimal conditions for each master mix may diverge and the conditions for one may not work well for the other. If this is the case, an independent optimization should be made for TaqDog 2x Green Master Mix.
Q2 – Should I inactivate the hot start element by an initial denaturation step?
No. TaqDog 2x Green Hot Start Master Mix uses an aptamer-based hot start technology. This element will efficiently disassociate as the first denaturation step is reached during the normal part of the thermal cycling program.
Q3 – Do I need to make any changes to my PCR protocol if I need to use the Enhancer Reagent?
Likely yes. Commonly, optimization of the annealing temperature may be required when using the Enhancer Reagent. It is often sufficient to simply reduce the annealing temperature by 2 – 3°C. If still no band is visible after adjusting by 2 – 3°C, 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 7°C below this temperature. If primers have different melting temperatures use the lower of the two melting temperatures as reference.
Q4 – Do I need to optimize annealing temperature when using TaqDog 2x Green Master Mix?
In general, no. For new protocol development, always first optimize conditions by running at several annealing temperatures. The temperatures tested should range from the predicted melting temperature of the primer all the way down to 7°C below the melting temperature. If primers have different melting temperatures use the lower of the two melting temperatures as reference.
Q5 – Do I need to optimize the number of cycles for PCR when using TaqDog 2x Green Master Mixes?
In general, no. You should always start at an expected/anticipated number of cycles. If this doesn’t work, then you should increase the number of cycles by steps of 3. TaqDog 2x Green Master Mixes are effective between 25 and 40 cycles.
Q6 – Why doesn’t TaqDog 2x Green Master Mix work when I use my existing PCR protocol?
There are four likely reasons:
i) Your PCR conditions require the addition of the PCR Enhancer Reagent to help overcome inefficient amplification. You should empirically determine the optimal amount of PCR enhancer to use by titrating the amount added in increments of 5 µl, up to a maximum of 20 µl (In a 50 µl PCR reaction).
ii) 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°C. If still no band is visible after adjusting by 7°C, 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 7°C below this temperature. If primers have different melting temperatures use the lower of the two melting temperatures as reference.
iii) 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 95°C. 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 94°C for the number of cycles required to amplify the product.
iv) If no band is visible, please increase the number of cycles for the PCR protocol by increments of 3. TaqDog 2x Green Master Mixes are effective between 25 and 40 cycles.
Q7 – Are TaqDog 2x Green Master Mixes appropriate to use with bacterial PCR?
In general, no. This fraction of TaqDog recombinant polymerase is purified from E. coli. A low level of residual E. coli DNA is present, so depending upon the homology of the bacterial species in question, it might be advised to instead use an ultra-pure version of Taq polymerase that is guaranteed to be free of bacterial DNA.
Q8 – How many times can I freeze-thaw TaqDog 2x Green Master Mixes?
TaqDog 2x Green Master Mixes have shown no appreciable loss of activity after 10 or more freeze-thaw cycles in our own testing.