Hi Lindsay,

We used the Phusion High Fidelity DNA Polymerase (NEB) successful for many years in our lab. The performance of the Phusion High Fidelity Polymerase (Thermo Fisher) was almost the same. However, the Q5 High-Fidelity DNA Polymerase (NEB) seems to be even better. I used this enzyme for several PCRs prior to cloning. All these polymerases have a proof reading activity. You should not use a Taq without it if PCR errors are an issue.

You can use the Q5 master mix if you have enough money. It saves some time. Nevertheless, mixing dNTPs with buffer and enzyme is easy.

Good luck with your experiment!

Boas 

Dear Lindsay 

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Hi Lindsay,

We used the Phusion High Fidelity DNA Polymerase (NEB) successful for many years in our lab. The performance of the Phusion High Fidelity Polymerase (Thermo Fisher) was almost the same. However, the Q5 High-Fidelity DNA Polymerase (NEB) seems to be even better. I used this enzyme for several PCRs prior to cloning. All these polymerases have a proof reading activity. You should not use a Taq without it if PCR errors are an issue.

You can use the Q5 master mix if you have enough money. It saves some time. Nevertheless, mixing dNTPs with buffer and enzyme is easy.

Good luck with your experiment!

Boas 

Hi Lindsay, 

I personally like Q5, i work with human samples and i need a high fidelity Taq. The only thing to consider is that with Q5 the Ta is usually higher than with a normal PCR and that sometimes you need to add DMSO to your PCR mix. Other than that, i highly recommed Q5. 

Good Luck 

Thanks everyone for your advice. I've worked out that using the Q5 Hot-Start + buying dNTPs will cost a total of £686 for my samples. Using the Phusion Hot-Start Mastermix will be £747. So a difference of £61, which isn't much. Ideally I'd use Q5 Hot-start mastermix but as far as I can see, this doesn't exist. You can only buy it together with buffer but no dNTPs. Or as a mastermix but not being a hot-start. How much better do you think the hot-start option would be?

The question is....

Would I be better off using the Phusion Mastermix for easiness and to avoid more pipetting contamination/errors, OR would it be better to use the Q5 which is arguably the better enzyme, even though I'd have to mix enzyme, buffer and dNTPs? OR forget the hot-start and get the Q5 mastermix? What would you do?!

Thanks!

Hi Lindsay,

you should not worry about pipetting errors if you just have to add dNTPs and buffer. Differences in the product amount will occur due to different primers/templates and different concentrations of primers/templates. You have to add primers and templates and pipetting errors will occur at that point. The effect of slightly different dNTP concentrations is not important as long as there are enough in the reaction. Therefore, I would recommend using the Q5.

However, I never observed any difference between the Hot-Start and the classic version of polymerases. You can prepare reactions on ice to minimize the enzyme activity prior to program start.

Good luck!

Thank you Boas.

I will go for the Q5 I think. I was more worried about contamination as someone else in my lab has had problems with contaminated primers, so I wanted to avoid too much pipetting but realistically two more steps probably isn't going to make any difference.

Many thanks for your help!

Hello Lindsay,

I tested different high fidelity enzymes for other purposes, included cloning of big chunks of DNA. Personally, I definitely switched to Q5 master mix: since then, never had any problem of mistakes inserted in my sequencing and, let's say, 2X master mix is so much easier to use. I would not worry about the hot start features: I normally set my PCR at RT to stabilize all the buffers and no problem (they suggest to totally thaw it to eliminate the eventual precipitate).

In conclusion, Q5 2x master mix is my first choice for precision and versatility.