Hi Himanshi,

I am not using Fusion Taq, so I can´t compare, but I have the best results with Hot Start Taq.

Good luck.

Lenka

Cloning experiments require amplicons with high fidelity and lowest errors in sequence. I would chose Fusion polymerase.

I'm pretty sure Fusion Taq results in blunt ends? --> so if you are going to go with it make sure you purchase the correct cloning 'kit' (i.e. not ones that requires 'sticky' 'A overhangs for insertion into vector)

Phusion for sure because of the high fidelity and of the more efficient amplification of larger PCR products. In order to clone the product I would introduce restriction sites in the primer (preceded by the unspecific bases). Example: CCCGAATTCacgatcgatcgta - where the non-annealing bases including an EcoRI site are capped. A similar approach should be employed for the reverse primer - preferably with a different restriction site, but that depends on the vector into which you want to clone the product.

Ps: Yes, the Phusion does not yield any A overhangs for TA cloning.

Hi Himanshi,

I suppose you are talking about Phusion High fidelity by NEB. This would be your choice for clonig purposes since it has proof reading activity ensuring your product will be copied with a better fidelity than regular Taq polymerase, and in my experience, it produces acceptable PCR yield. Hot start enzymes are regular Taq pol wich have been manipulated to be activated at high temperatures, therefore improving the specificity of you reaction by avoiding so much mispriming at low temperatures. If not otherwise disclosed, they are not proofreading enzyme (i.e. they do not have 3'-5' exonucleolytic activity). Indeed, if you use one of the several high fidelity enzymes, such as Phusion, the exonuclease activity will trimm the A overhang typical of Taq pol PCR products. As the colleagues have said before, you will then have either to (i) use a "blunt" kit for cloning; (ii) design your primers with restriction sites in order to digest with specific enzyme and then ligate with (f.i.) T4 ligase, or (iii) use a TA cloning system, then you'll have to adenilate your PCR products (fresh) with a simple reaction consisting of 4 uL dATP (10 mM), 1 uL MgCl2 (50 mM), 1 U Taq polymerase (regular) and 40 ul of your PCR product (you can scale up or down a piacere and also use purified PCR products), 1 h at 72 ºC followed by column purification or precipitation. I advise you to use the adenilated product in a week period, since A tails use to be spontanously trimmed).

Hope to have been useful. Good luck!

Rocio