I partially disagree with the responses above, because it depends upon how you are doing the sequencing. If you are going to clone the PCR product and then sequence individual clones, then yes you should use high fidelity polymerase. However if you are going to directly sequence the PCR product itself without cloning, then there is no need to use high fidelity polymerase. The reason is that with a cloning step, you are sequencing from one individual molecule. But if you sequence the PCR end product directly, it is a population of molecules and the overall population should not show the mutations.

You'll thank yourself later if you use the high-fidelity master mix.  It will greatly reduce the likelihood of errors in your amplified sequence.

Of course, It's a better idea to use a Hi-Fi (Hi-Fidelity) master mix if you want to avoid error in your PCR product. Best wishes!

Hi there,

your point is to get sure about the sequence therefore you have to use the more accurate method. Go for HF!

 If you want to persuade yourself run the error rate calculator below:

https://www.thermofisher.com/fr/fr/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/pcr-fidelity-calculator.html#

I partially disagree with the responses above, because it depends upon how you are doing the sequencing. If you are going to clone the PCR product and then sequence individual clones, then yes you should use high fidelity polymerase. However if you are going to directly sequence the PCR product itself without cloning, then there is no need to use high fidelity polymerase. The reason is that with a cloning step, you are sequencing from one individual molecule. But if you sequence the PCR end product directly, it is a population of molecules and the overall population should not show the mutations.

Well I don't exactely get Michael's point if the purpose of the experiment as exposed in the initial question is to actually establish/read a sequence. I don't see the point of reading a sequence in which one could find errors/ambiguities due to the lack of fidelity of the polymerase where the simple use of HF enzyme would give a much clearer sequence...

Hello Dominique Liger.

well , i think  Michael want to say :

that the reading intensity in sanger sequencing machine depend on the collection of signals which come from a population of templates( a huge number of template ) , and the error rate of Taq polymerase is about 1/9000 in each nucleotide , so this error signal will not effect the final intensity of reading signal and will not effect the accuracy of sequence reading.

Right, I get it now... Cheers!

Thanks Hayder - you explained my answer better than I had.