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Q1 – Should I inactivate the hot start element by an initial denaturation step?
No. TaqDog Hot Start Polymerases 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.
Q2 – 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.
Q3 – Do I need to optimize annealing temperature when using TaqDog Polymerases?
In general, no. In the case of 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.
Q4 – Do I need to optimize the number of cycles for PCR when using TaqDog Polymerases?
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 Polymerases are effective between 25 and 40 cycles.
Q5 – Are TaqDog polymerases 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.