56
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
scriptase, DNA polymerase, or terminal transferase-catalyzed reactions.
Two sequential precipitations result in the removal of 99% of the dNTPs
and greater than 90% DN A recovery ( Okayama and Berg 1982 ). However,
ammonium acetate should not be used, when the precipitated nucleic
acid is to be phosphorylated or tailed, since ammonium ions inhibit
the enzymes required for these processes.
Li Cl (0.8 M, final concentration) is frequently used for precipitation at70°C, since LiCl is very soluble under this condition. Li+ should be
avoided when the precipitated RNA is to be reverse transcribed. No
chloride should be used when translation in cell-free systems is intended,
since ci- interferes with the initiation of protein synthesis. Should the
sample contain SDS, sodium chloride (0.2 M) should be used. The detergent then remains soluble in 70% ethanol. Sampies containing phosphate or 10 mM EDTA should be dialysed prior to precipitation, as these
materials will precipitate along with the nucleic acid.
• Isopropanol is useful forprecipitation of nucleic acids, if smallervolume of
alcohol are needed, e.g., when precipitating DNA or RNA from volumes
>0.5 ml in a microcentrifuge tube. Substitute 1 vol isopropanol for 2.5 vol
100% ethanol. But salts are less soluble in isopropanol than in ethanol.
Subprotocol 2
Isolation of Genomic DNA From Mammalian Cells
Principles of DNA isolation. Genomic DNA is mainly used for restriction
enzyme digestion and mutation analysis, Southern blots, PCR and library
construction. Isolation from tissue culture cells is particularly convenient,
but preparation from blood or whole organs is also possible. The major
difficulties encountered during preparation are the high viscosity of genarnie DNA, which makes it difficult to redissolve after alcohol precipitation
and its high-molecular weight (average human chromosome: 100-150 Mb)
which makes it sensitive to shear forces (Birnboim 1992). Care should also
be taken to avoid any contamination by recombinant DNA. A variety of
commerically available purification kits exists (e.g. Qiagen®). But there
are also rather easy laboratory protocols. The one provided here, allows
for preparation of genomic DNA from different sources, i.e. cultured cells,
tissue samples or blood samples. (Recently, a single-tube genomic DNA isolation method from blood was reported by Gruberand Zingales, 1995.)
The DN Ais isolated by lysing cells in the presence ofhigh concentration of
EDTA ( chelates divalent cations which are essential for the action of most
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
scriptase, DNA polymerase, or terminal transferase-catalyzed reactions.
Two sequential precipitations result in the removal of 99% of the dNTPs
and greater than 90% DN A recovery ( Okayama and Berg 1982 ). However,
ammonium acetate should not be used, when the precipitated nucleic
acid is to be phosphorylated or tailed, since ammonium ions inhibit
the enzymes required for these processes.
Li Cl (0.8 M, final concentration) is frequently used for precipitation at70°C, since LiCl is very soluble under this condition. Li+ should be
avoided when the precipitated RNA is to be reverse transcribed. No
chloride should be used when translation in cell-free systems is intended,
since ci- interferes with the initiation of protein synthesis. Should the
sample contain SDS, sodium chloride (0.2 M) should be used. The detergent then remains soluble in 70% ethanol. Sampies containing phosphate or 10 mM EDTA should be dialysed prior to precipitation, as these
materials will precipitate along with the nucleic acid.
• Isopropanol is useful forprecipitation of nucleic acids, if smallervolume of
alcohol are needed, e.g., when precipitating DNA or RNA from volumes
>0.5 ml in a microcentrifuge tube. Substitute 1 vol isopropanol for 2.5 vol
100% ethanol. But salts are less soluble in isopropanol than in ethanol.
Subprotocol 2
Isolation of Genomic DNA From Mammalian Cells
Principles of DNA isolation. Genomic DNA is mainly used for restriction
enzyme digestion and mutation analysis, Southern blots, PCR and library
construction. Isolation from tissue culture cells is particularly convenient,
but preparation from blood or whole organs is also possible. The major
difficulties encountered during preparation are the high viscosity of genarnie DNA, which makes it difficult to redissolve after alcohol precipitation
and its high-molecular weight (average human chromosome: 100-150 Mb)
which makes it sensitive to shear forces (Birnboim 1992). Care should also
be taken to avoid any contamination by recombinant DNA. A variety of
commerically available purification kits exists (e.g. Qiagen®). But there
are also rather easy laboratory protocols. The one provided here, allows
for preparation of genomic DNA from different sources, i.e. cultured cells,
tissue samples or blood samples. (Recently, a single-tube genomic DNA isolation method from blood was reported by Gruberand Zingales, 1995.)
The DN Ais isolated by lysing cells in the presence ofhigh concentration of
EDTA ( chelates divalent cations which are essential for the action of most