hmm? difficult question

I small additives are interfering with cleanup then possibly just a quick spin of the amplimer through size exclusion matrix like Sephadex will take out all of the small molecules and then do the clean up may be a quicker option than working up a new polymerase or even purifying out of agarose gel electrophoresis

Paul Rutland Paul, thank you for your answer! Yes, agarose gel extraction is not effective because I need about 1 mkg of the amplicon. I think these additives are something like carbohydrates. They don't clean out during ethanol precipitation and even persist after magnetic beads clean up. Magnetic beads behave very strangely. They stick together. But anyway some products elute. The eluted solution seems viscous and foamy. And when I tried cleaning products on silica membrane columns I lost almost everything((

You can try SeqAmp DNA Polymerase (Takara) or

PrimeSTAR Max DNA Polymerase (Takara)

Good Luck

Liubov Skorodumova if carbohydrate is the problem perhaps CTAB dna precipitation may help ( it seems to be the method of choice for plant geneticists) as etoh does not. Perhaps also the contaminant is a detergent at too high concentration or too low temperature forming micelles. Tech help at Isogene probably will not tell you the buffer constituents but they may have some insight into post pcr purification.I have always found tech supports really helpful

You could try Promega GoTaq™ Long PCR Master Mix which contains the high-performance GoTaq® Hot Start Polymerase. Also, PrimeSTAR HS DNA Polymerase (developed by Takara Bio) is recommended. Both are high-fidelity DNA polymerases with amplification of long amplicons.

Paul Rutland , CTAB buffer precipitation worked well! Contaminants went away and amplicons weren't viscous at all.

Rajwali Khan , Zohreh Moradi , thank you for your recommendations!

I will keep in mind these polymerases and we will order them as soon as possible.

After all, I tried LongAmp from NEB (colleague gave a sample) and it worked well.

It's very interesting because Q5 High-Fidelity DNA Polymerase (NEB) didn't amplify this product even with GC enhancer...

Excellent well done Liubov Skorodumova .Thank you for the update

hmm? difficult question

I just published a simple, detailed protocol for long-range PCR (up to 20kb in size) for targeted sequencing. I used the LA Taq Polymerase from TAKARA Bio.

Check it out:

Preprint Long-range PCR-based NGS applications to diagnose Mendelian ...