Posted on May 16th, 2016 by UNM CC
(Ref: Molecular Cloning A8.12)
The major considerations in using alcohol to precipitate DNA are:
- Temperature: -20°C is optimal, but 0°C can be used for >20 ng/mL
- 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.
- Time: Optimal precipitation requires >1 hr at -20°C.
- 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.
- Mg++ [10 uM] helps pellet oligonucleotides (<100 bp). Otherwise Mg should be avoided because it may activate DNAses.
- 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.
- 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.
- 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.