I am working on amplifying larger fragments (20.5 kb & 22.5 kb) using Phusion Polymerase and Q5 Polymerase. I am unsing isolated genomic DNA of Corynebacterium glutamicum as template. I am facing difficulty in getting pure desired fragment instead I am getting side products as well of around 6 kb, 3kb and 1 kb. How to get rid of these side products?

I am using these fragments alongwith two other fragments to ligate them together using NEB HiFi DNA assembly mix.

1) 1.1 kb fragment

2) 4.4 kb fragment

3) 22.5 kb fragment

4) 20.5 kb fragment

The problem I am facing is when I take all these 4 fragments alongwith assembly mix in 20 µl reraction mix I have total DNA 0.012 pmoles where as reccomended range of total DNA by NEB for ligating 4-6 fragments is 0.2-0.5 pmoles in 20 µl reaction.

My Gibson assembly is not working. Is this due to lower total pmoles of DNA or due to other impurities or side products.

I have also tried extracting desired product from the gel, but the concentration is low and it has impurities like protein and salts. When I tried to remove these impurities using phenol-chloroform extraction, my DNA concentration was in negatives (measured by Nanodrop)

If you have any idea where I am making mistakes please let me know.