for the PCR I use the Phusion polymerse from NEB rather than Pfu or Taq... Using Pfu and Taq the annealing Temp is 5- 10 grade lower than the calculated Tm, but what about the Phusion polymerse, should I use higher annealing temp???
Thanks a lot for your answers... I tested different annealing tempretures starting from the the one which I got calculated from the WEBSITE (https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator#.UIm8BMXO1K0) which is uded for the calculation of the annealing temp for Phusion polymerase. [Results: The high annealing Temp didn´t produce any product )
The best result I got, is when using the classical annealing temp, (I mean Tm - 5 ° C).
Yes, you are supposed to use the annealing temperature given by the NEB website for the phusion polymerase.
Here is the link:
https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator#.UIm8BMXO1K0
Feed the enzyme (phusion), forward and reverse primers and then click calculate.
It is a good enzyme to work on, and for better results addition of DMSO and optimizing the Mg2+ concentrations can be helpful.
The phusion polymerase tolerates a wide array of annealing temperatures so you should probably choose an annealing temperature that is specific to the primers you are using to ensure you don't get non-specific products. When you've calculated (or some companies provide) the annealing temperature of your primer pair, I suggest you do an annealing gradient where you test temperatures 5-10 degrees below and above the recommend annealing temperature. This will allow you to assess the best temperature for optimal yield of your product of interest and minimal non-specific products. The enzyme itself is quite stable and should work fine at most temperatures - it is most important to use the manufacturer's temperature for the elongation step, which is when the polymerase is active. I believe this temperature is 72'C for NEB phusion so annealing temperatures between 40-72 should all work (lower annealing gives more non-specific products).
Phusion is a very tolerant polymerase. Even with your calculated Tm, this is just a guide for a starting point. Anyway, you should try and if you don't get good amplification a gradient is the easiest solution.
Thanks a lot for your answers... I tested different annealing tempretures starting from the the one which I got calculated from the WEBSITE (https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator#.UIm8BMXO1K0) which is uded for the calculation of the annealing temp for Phusion polymerase. [Results: The high annealing Temp didn´t produce any product )
The best result I got, is when using the classical annealing temp, (I mean Tm - 5 ° C).
Is it ideal to use Herculase II Phusion polymerase (Agilent) if I want to amplify a 7kb segment with a vector overhang ?Or can you please suggest me which enzyme can be used to get a 7kb long segment with an overhang?
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