I am working on a project what requires the DNA polymerase to copy a long stretch of DNA ( up to 6kb).
One of the most important options I need is its proofreading activity.my construction shouldn't have been mutated but unfortunately I am not in a good shape to prepare an specific Enzyme like pwo
and the other problem is I should have a blunt end insert.
can I amplify my insert with a mix formulation of taq and pfu? if so how I can do it?
if not, what is a good candidate DNA polymerase that can do the trick?
thank you very much indeed for your time
I agree with Lori and Wael, Phusion is a good choice from my experience.
Thermo Scientific works with an High fidelity DNA Polimerase, that enables the use of extremely short PCR protocols (15 s/1 kb) with both low and high complexity DNA templates. In my experience, this Master Mix can amplify everything in short time.
https://www.thermofisher.com/pe/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-molecular-biology-products/phusion.html
Except for DNA polymerase, other conditions also can affects your results. Such as primer, Template concentration and purity, annealing temperature etc. Because the copied DNA up to 6kb, so some conditions you should have a try.
Yes, design long primer (30bp, with high Tm), use higher annealing temperature, actually you only need 2 steps - denature and extension (1min per kb). Elongase can amplify up to 15kb.
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