May 16, 2016

Alcohol Precipitation of DNA


(Ref: Molecular Cloning A8.12)

The major considerations in using alcohol to precipitate DNA are:

  1. Temperature: -20°C is optimal, but 0°C can be used for >20 ng/mL
  2. Amount: For small amount of DNA (<100 ng, i.e. too little to reliably see a pellet) the use of glycogen (1 µL of a 20 mg/mL stock, (Roche 10901393001) will increase yield and allow visualization of the pellet.
  3. Time: Optimal precipitation requires >1 hr at -20°C.
  4. Speed of centrifuge: High speeds are important for small amounts or small oligos. Typically they should be spun at >12,000 rpm (> 12,000 g) in a microcentrifuge.
  5. Mg++ [10 uM] helps pellet oligonucleotides (<100 bp). Otherwise Mg should be avoided because it may activate DNAses.
  6. Avoid co-precipitates: EDTA (>10 mM) and Ca (>1 mM) will precipitate at the concentrations indicated in alcohol solutions. SDS will also precipitate when a salt other than NaCl is employed.
  7. Choice of alcohol: Isopropanol has the advantage of requiring less volume. Typically 0.6 vol to 1 vol. of isopropanol is added to 1 volume of DNA/salt soulution. The higher amount is useful for smaller RNA fragments. In contrast, 2 volumes of ethanol is typical.) However, isopropanol has the disadvantage of coprecipitating more salts and is less volatile (so it is slow to air dry) compared to ethanol.
  8. Choice of salt:

NaOAc, pH5.2, [0.3M]: This is the standard. (10x Stock concentration = 3 M)

NaCl [0.2M]: Useful if SDS is included because it is more soluable in NaCl (Stock conentrations vary 1M - 5M)

NH4OAc [2 - 2.5M]: Less coprecipitation of dNTPs, good for purifying oligos. However, NH4+ ions inhibit polynucleotide kinase).

LiCl [0.8M]: Soluble in a higher concentration of ethanol which is useful for the precipitation of RNA. However Cl- inhibits the initiation of protein synthesis and RNA will display varying solubility based on size.

Tags: Fero-DNA, Fero-Lab-Protocols