Feb 27, 2023

Ethanol precipitation of nucleic acid

This protocol is a draft, published without a DOI.
  • 1University of Oslo
Andreas Sagen
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Protocol Citation: Andreas Sagen 2023. Ethanol precipitation of nucleic acid. protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 11, 2023
Last Modified: February 27, 2023
Protocol  Integer ID: 76806
Keywords: ethanol precipitation of nucleic acid, ethanol precipitation, nucleic acid
Abstract
Simple protocol to precipitate nucleic acids
Protocol materials
GlycogenMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1767
Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2889
Acetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6283
Sodium acetate (3 M, pH 5.2)
Add 25 mL distilled water to a 50 mL canonical tube
Measure 12.305 g Sodium acetate

Materials:
Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2889
Adjust pH to 5.2 with glacial acetic acid

Materials:
Acetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6283
Autoclave solution at 121 °C for 00:15:00
15m
Precipitation
2h
Add 100 µL lysate in a reaction tube
Add 10 µL Sodium acetate (3 Mass Percent , 5.2 ), 2 µL 250 µg/mL glycogen and 333 µL absolute ethanol

Material:
GlycogenMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1767
Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2889
Vortex solution, then precipitate at -20 °C or -80 °C for 01:00:00 or Overnight

Note
Longer incubation and lower temperature provide better recovery of nucleic acids.

1h
Centrifuge at 12000 rcf, 4°C, 00:30:00 . Discard supernatant, and repeat step 9.1 and 9.2, 2 times in total
30m
Discard supernatant, and resuspend pellet in 500 µL 70% absolute ethanol
Centrifuge at 12000 rcf, 4°C, 00:30:00
30m
Air-dry the pellet

Note
Avoid over-drying the pellet, as it then takes more time to dissolve.

Resuspend pellet in 20 µL TE-buffer or nuclease-free water