Hi there,

Phusion has both high fidelity (error rate is less than 5.10-7) and high processivity (15-30s per kb) for amplicon up to 20kb . And you can get it at a very competitive price from ThermoFisher.

Hi there,

Phusion has both high fidelity (error rate is less than 5.10-7) and high processivity (15-30s per kb) for amplicon up to 20kb . And you can get it at a very competitive price from ThermoFisher.

Hi, for my experiments I am using Advantage polymerase mix from Clontech.

Phusion or Q5 polymerase, both are Hi Fidelity and can amplify more than 10kb. I used it always and no problems encountered.

Hi Priyanka, Use Takara Ex-Taq its a high fidelity enzyme for amplicon up to 20kb. I'm using it and getting satisfactory results. Good luck!!!

Hi,

I also recommend Phusion (R). Indeed, I don't want to optimize protocols on the one hand for a cheap Taq and a proof-reading Taq on the other hand. Therefore, I use Phusion nearly for all PCR experiments.

Regards.

Hi there,

The amplification that you are looking for can be done with almost any taq with a good fidelity and processivity, however, it depends on your downstream work that excatly what you want to do with that insert. If you are using it for protein expression, you should use a taq with high fidelity since a muation here and there screws up your tertairy structure of your protein. In any even, it is advisable to good a high fidelity taq since you do not want suprises down the tumoultous road of scientific resaerch.

Hi Priyanka,

1. If you only wish to use one of the enzymes you have in hand (listed above) and don't want to buy any more. You can go online to check out which one of these is 'High-Fidelity' and 'a long-range' DNA polymerase (such as can amplify up to 10kb etc).

2. Which additives? If you wish you can add 2-4% DMSO in case your template is G-C rich and prone to form a secondary structure

any high fidelity taq polymerase enzyme can work well for 3kb fragment. no worries. try any of the above suggested ones.

good luck

Regarding the additives in the PCR reaction (one of your questions):

Except DMSO, some people also use BSA to increase the specificity and efficiency of PCR performance. Yesterday, an article published by BioTechniques shows that another novel additive, Bovine Thrombin (BT), is more effective than one of the most popular additives, BSA. It indicated that BT was also able to effectively relieve PCR inhibition by nanomaterial inhibitors such as gold nanoparticles (AuNPs). See attached paper for more info.  

Hi Priyanka,

If you wish to use only one of the 7 DNA polymerases in your hand, you can choose the one based on (1) product length, (2) High-Fidelity, (3) Hot start, and (4) containing G-C rich formulated buffer. I went online to quickly check all of them, so:

1. The Platinum Master is out because it only amplifies up to 2.5 kb ( you have 3-kb); the Kapa amplifies up to 3.5-kb (very close to your product), I will hold this one until later; the Brazilian Taq cannot be found online.

2. Between AccuPrime GC and AccuPrime Taq (both amplify to 5 kb).The GC-rich formulated one should be better just in case your template is GC rich. Between NEB OneTaq Hot Start and NEB OneTaq (both amplify template up to 6 kb). Hot Start one is definitely better for PCR accuracy.

3. Finally, it comes to AccuPrime GC and NEB OneTaq Hot Start: both are Hot-start, both use GC-rich fomulated buffer; however, OneTaq Hot Start amplifies up to 6 kb, the later amplifies up to 5 kb. The OneTaq Hot Start also mixes with Deep VentR™ DNA polymerase to increase Fidelity.

4. So, I would try NEB OneTaq Hot Start DNA polymerase first to amplify your 3-kb product.

Hi Yuan,

Thank You so much for your answer... It was very detailed and helpful. 

Thanks everyone.,... all your answers helped me a lot.