3 Purification of Nucleic Acids From Eukaryotic Cells
55
when dealing with RNA) and as quickly as possible. Extractions are best
carried out on ice with refrigerated buffers to minimize nuclease activity.
Onlywhen SDS (see below) is present would a higher temperature (1 0 °C)
be required to prevent precipitation of the detergent.
• Purity
Detergents are employed to promote the dissociation of proteins from
nucleic acids. The most commonly used are the ionic detergents sodium
dodecylsulfate (SDS) (0.1 to 2o/o) or sodium desoxycholate, which in addition enhances the dissolution of lipid membrane structures (W allace
1987). The dissociation can furtherbe improved by incubation of the
sample with proteinase K (nuclease free) (50-200 Jlg/ml, final concentration; 37 oc for 30 min) in the presence ofSDS. To increase RNA over DNA
content, solutions can be made to pH 5-6. DNA is then selectively retained in the organic phase, leaving RNA in the aqueous phase. The
low pH also reduces the activity of many nucleases.
• Yield
For maximum recovery, re-extract all organic phases by adding 100 111 of
TE-buffer to the first organic phase and proceed with this aqueous phase
through all subsequent organic phases. Then combine this second aqueous phase with the first and concentrate by preciptation.
After 1-15 min at 0 oc in the absence of carrier, nucleic acid concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively
recovered by centrifugation in a microcentrifuge (Sambrock et al. 1989).
Torecover nanogram quantities of nucleic acids, add 10 Jlg of 10 Jlg/Jll
carrier tRNA prior to extraction. If this is not possible due to interference
with subsequent experiments, add 0.1 vol of 1 M spermine tetrahydrochloride (Hoopes and McClure 1981) or apply more rigorous ultracentrifugation like in the technique of Shapiro (1981). To short DNA
fragments (2 to 10 mM final concentration), increase ice-cold ethanol
to 3 vol, and precipitate at -70 oc for 30-60 min. Large volumes or nucleic
acid samples of unknown concentration should also be precipitated for
30-60 min at -70 oc to permit temperature equilibration to obtain a maximum precipitate.
• Precipitation
The choice of salt used for precipitation is determined both by the nature
of the sample and by the intended use of the nucleic acid. Substitution of
sodium acetate (pH 5.2) by ammonium acetate (2.0-2.5 M, final concentration) prevents the precipitation of dNTPs and therefore is useful for
removing unreacted triphosphates from the products of reverse tran-
55
when dealing with RNA) and as quickly as possible. Extractions are best
carried out on ice with refrigerated buffers to minimize nuclease activity.
Onlywhen SDS (see below) is present would a higher temperature (1 0 °C)
be required to prevent precipitation of the detergent.
• Purity
Detergents are employed to promote the dissociation of proteins from
nucleic acids. The most commonly used are the ionic detergents sodium
dodecylsulfate (SDS) (0.1 to 2o/o) or sodium desoxycholate, which in addition enhances the dissolution of lipid membrane structures (W allace
1987). The dissociation can furtherbe improved by incubation of the
sample with proteinase K (nuclease free) (50-200 Jlg/ml, final concentration; 37 oc for 30 min) in the presence ofSDS. To increase RNA over DNA
content, solutions can be made to pH 5-6. DNA is then selectively retained in the organic phase, leaving RNA in the aqueous phase. The
low pH also reduces the activity of many nucleases.
• Yield
For maximum recovery, re-extract all organic phases by adding 100 111 of
TE-buffer to the first organic phase and proceed with this aqueous phase
through all subsequent organic phases. Then combine this second aqueous phase with the first and concentrate by preciptation.
After 1-15 min at 0 oc in the absence of carrier, nucleic acid concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively
recovered by centrifugation in a microcentrifuge (Sambrock et al. 1989).
Torecover nanogram quantities of nucleic acids, add 10 Jlg of 10 Jlg/Jll
carrier tRNA prior to extraction. If this is not possible due to interference
with subsequent experiments, add 0.1 vol of 1 M spermine tetrahydrochloride (Hoopes and McClure 1981) or apply more rigorous ultracentrifugation like in the technique of Shapiro (1981). To short DNA
fragments (2 to 10 mM final concentration), increase ice-cold ethanol
to 3 vol, and precipitate at -70 oc for 30-60 min. Large volumes or nucleic
acid samples of unknown concentration should also be precipitated for
30-60 min at -70 oc to permit temperature equilibration to obtain a maximum precipitate.
• Precipitation
The choice of salt used for precipitation is determined both by the nature
of the sample and by the intended use of the nucleic acid. Substitution of
sodium acetate (pH 5.2) by ammonium acetate (2.0-2.5 M, final concentration) prevents the precipitation of dNTPs and therefore is useful for
removing unreacted triphosphates from the products of reverse tran-