Does this make a difference to the outcome of the PCR?
The initial denaturation has two purposes if you buy the phusion hot start.
The antibody or aptamer which is bound to phusion at low temperatures needs to be dissociated from your polymerase to allow it to bind and extend DNA. In addition, the initial heat also denatures your template fully.
If you do not have the hot start and are only using this initial heating to denature your template, 95 or 98 will not make any difference. Very few templates will require a melting temperature above 95 and you would probably know it if you were working on it.
In summary, 95 will be fine unless you have phusion hot start, as the aptamer/antibody binding is finely tuned and you need 98 to knock it off.
Any particular reason you want to do 95? Or is it because you have polymerases that use 95 so all your programs use it? :D
Initial denaturation is totally depend on your template. if it is having High G+C content you have to do either increase Initial denaturation temperature to a maximum that polymerase can tolerate or with a temperature ranging from 92-96 for long time) Due to the high thermostability of Phusion DNA Polymerase even higher than 98°C denaturation.
I know with KAPA HiFi phusion it is recommended that the innitial denaturation be at 95 for 2-5 min. We have used this and it worked always. On the other hand Phsion Hot start requires higher temeperatures such as 98 degress but again it will depend on the template.
Again, it depends on your template, but an initial denaturation for 5 minutes at 95 degree will open mostly template...so to answer...no, no difference at all in my experience.
The initial denaturation has two purposes if you buy the phusion hot start.
The antibody or aptamer which is bound to phusion at low temperatures needs to be dissociated from your polymerase to allow it to bind and extend DNA. In addition, the initial heat also denatures your template fully.
If you do not have the hot start and are only using this initial heating to denature your template, 95 or 98 will not make any difference. Very few templates will require a melting temperature above 95 and you would probably know it if you were working on it.
In summary, 95 will be fine unless you have phusion hot start, as the aptamer/antibody binding is finely tuned and you need 98 to knock it off.
Any particular reason you want to do 95? Or is it because you have polymerases that use 95 so all your programs use it? :D
I think initial denaturation for phusion polymerase 98oC is best one
The reason for 98°C denaturation with Phusion is not because to secure knocking off the affibody present in the Hot Start version. The affibody will separate already above 50°C and binding ability is in fact reversible (unlike antibody based versions). The reason for the recommended high denaturation temperature is that the buffer has a high salt concentration and consequently GC rich templates may not denature sufficiently at 95°C. For the same reason higher annealing temperatures are recommended with Phusion. Nevertheless, 95°C should work unless your template is very GC rich. Phusion is very heat stable and thus recommending 98°C is a simple solution as it should work for almost any target.
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