UPDATE: I sent to sequence directly after ethanol precipitation mentioned below and I got very good quality of reads (I precise that I had only one clear band)

I want to Sanger sequence a 500 bp amplicon and I do not have any PCR Cleanup kit or Gel extraction kit left. Do someone have experience with Ethanol precipitation for this application?

The classical precipitation using:

- PCR reaction

- 0.1 vol of 3M Sodium Acetate

- 2-3 Vol of 100% EtOH

+ Pellet wash with 70% EtOh

Gives me a good recovery but I'm concerned about remaining oligos and dNTPs that could affect the sequencing reaction.

Is there a way to selectively precipitate >50-100 bp fragments by playing with the parameters (Salt, concentration, EtOH ratios..)? I've seen that Sodium Ammonium may be used instead of NaAc to exclude dNTPs, would the oligos precipitate as well? And may Sodium Ammonium inhibit the sequencing reaction?

https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/

I've also heard of selective size precipitation using PEG in the context of library prep but I guess my amplicon is too small for this approach.

PS: I use the Sanger sequencing service from Azenta/Genewiz.

Thank you for your Help!