- Perform an ethanol precipitation [1]:

  - Add 1/10 volume 3M sodium acetate, pH=5.2 or 
    other appropriate salt [2].

  - Add 2 volumes ice-cold ethanol and mix well.

  - If necessary, divide the sample between microfuge 
    tubes such that none holds more than 400 µL [3].

  - Incubate at 0°C for 15-30 min [4,5].

  - Centrifuge maximum speed, 10 min, 4°C [6] and 
    discard supernatant.

  - Add 800 µL 70% ethanol.

  - Centrifuge maximum speed, 10 min, 4°C [6] and 
    discard supernatant.
  
  - Leave tube open at room temperature until ethanol 
    has evaporated [7,8].

Notes:
[1] Sambrook2006: 10.1101/pdb.prot4456
    Green2016: 10.1101/pdb.prot093377

[2] 300 mM sodium acetate, pH 5.2: Used for most 
    routine precipitations of DNA and RNA.

    2M ammonium acetate: Used to reduce the 
    coprecipitation of unwanted contaminants (e.g.  
    dNTPs or oligosaccharides) or when nucleic acids 
    are precipitated after digestion of agarose gels 
    with agarase. It should not be used when the 
    precipitated nucleic acid is to be 
    phosphorylated.

    200 mM sodium chloride: Used if the DNA sample 
    contains SDS.

    800 mM lithium chloride: Used when high 
    concentrations of ethanol are required for 
    precipitation (e.g. large RNAs). 

[3] Alternatively, an ultracentrifuge can be used 
    instead of a table-top centrifuge.

[4] Usually 15-30 minutes is sufficient, but when the 
    size of the DNA is small (<100 nucleotides) or 
    when it is present in small amounts (<100 pg/µL), 
    extend the period of storage to at least 1 hour 
    and add MgCl₂ to a final concentration of 10 mM.

[5] DNA can be stored indefinitely in ethanolic 
    solutions at either 0°C or −20°C.

[6] Mark the edge of the tube so you can find the 
    pellet.  If low concentrations of DNA (<20 pg/µL) 
    or very small fragments (<100 nucleotides) are 
    being processed, more extensive centrifugation 
    may be required.  Centrifugation at 100,000g for 
    20-30 minutes allows the recovery of picogram 
    quantities of nucleic acid in the absence of 
    carrier.

[7] Do not dry pellets of nucleic acid in a 
    lyophilizer, as this causes denaturation of small 
    (<400-nucleotide) fragments of DNA and greatly 
    reduces the recovery of larger fragments of DNA. 

    If necessary, the open tube containing the 
    redissolved DNA can be incubated for 2-3 minutes 
    at 45°C in a heating block to allow any traces of 
    ethanol to evaporate.

[8] Up to 50% of the DNA is smeared on the wall of 
    the tube. To recover all of the DNA, push a bead 
    of fluid backward and forward over the 
    appropriate quadrant of wall with a pipette tip.

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