70
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
11. Spinat 15000 rpm for 15 min at 4°C.
12. Wash the pellet (which is often invisible) with 70% ethanol.
13. Redissolve the RNA in a small volume ofwater. Measure the absorbance
at 260 nm, using HCl:methanol (1:1) treated cuvettes.
Note: Approximately 1% of the input RNA should be recovered as poly(A)+
RNA. The yield of poly(A)+ RNA from 10 7 cultured mammalian cells should
be 1-5 J.lg.
14. Transfer the RNA to a polypropylene microcentrifuge tube, add 3 vol of
100% ice-cold ethanol, mix well, and store the preparation at -70°C. To
recover the RNA, add 3M sodium acetate (pH 5.2) to a final concentration of 0.3 M, mix well, and spin at 15000 rpm for 15 min at 4°C.
References
A viv R, Leder P (1972) Purification ofbiologically active globin messenger RN A by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sei USA 69:1408-1412
Birnboim RC (1992) Extraction ofhigh molecular weight RNA and DNA from cultured
mammalian cells. Methods Enzymol216:154-60
Brawerman G, Mendecki J, Lee SY (1972) A procedure for the isolation of mammalian
messenger ribonucleic acid. Biochemistry 11:637-641
Chomczynski P, Sacchi N (1987} Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Bioehern 162:156-159
Edmonds M, Vaughan Jr MR, Nakazato R (1971) Polyadenylic acid sequences in the
heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of ReLa cells:
possible evidence for a precursor relationship. Proc Natl Acad Sei USA 68:1336-1340
Gough NM (1988) Rapid and quantitative preparation of cytoplasmic RNA from small
numbers of cells. Analytic Biochem. 173:93-95
Gruber A and Zingales B (1995) One-base double-stranded DNA sequencing using commercial kits. BioTechniques 19:748-750
Roopes BC, McClure WR (1981) Sturlies on the selectivity ofDNA precipitation by spermine. Nucleic Acids Res 9:5493-5504
Liedke W, Battistini L, Brosnan CF, Raine CS (1994} PCR Methods Applic 3:185-187
Okayama R, Berg P (1982) Righ-efficiency cloning of full-length cDNA. Mol Cell Biol
2:161-170
Sambrook J, Fritsch, EF, Maniatis T ( 1989} Molecular cloning. A laboratory manual. Cold
Spring Rarbor Laboratory Press, Cold Spring Rarbor
Shapiro DJ (1981) Quantitativeethanol precipitation of nanogram quantities of DNA
and RNA. Analytical Bioehern 110:229-231
Wallace DM (1987} Large- and small-scale phenol extraction. Methods Enzymol 152:
33-41
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
11. Spinat 15000 rpm for 15 min at 4°C.
12. Wash the pellet (which is often invisible) with 70% ethanol.
13. Redissolve the RNA in a small volume ofwater. Measure the absorbance
at 260 nm, using HCl:methanol (1:1) treated cuvettes.
Note: Approximately 1% of the input RNA should be recovered as poly(A)+
RNA. The yield of poly(A)+ RNA from 10 7 cultured mammalian cells should
be 1-5 J.lg.
14. Transfer the RNA to a polypropylene microcentrifuge tube, add 3 vol of
100% ice-cold ethanol, mix well, and store the preparation at -70°C. To
recover the RNA, add 3M sodium acetate (pH 5.2) to a final concentration of 0.3 M, mix well, and spin at 15000 rpm for 15 min at 4°C.
References
A viv R, Leder P (1972) Purification ofbiologically active globin messenger RN A by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sei USA 69:1408-1412
Birnboim RC (1992) Extraction ofhigh molecular weight RNA and DNA from cultured
mammalian cells. Methods Enzymol216:154-60
Brawerman G, Mendecki J, Lee SY (1972) A procedure for the isolation of mammalian
messenger ribonucleic acid. Biochemistry 11:637-641
Chomczynski P, Sacchi N (1987} Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Bioehern 162:156-159
Edmonds M, Vaughan Jr MR, Nakazato R (1971) Polyadenylic acid sequences in the
heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of ReLa cells:
possible evidence for a precursor relationship. Proc Natl Acad Sei USA 68:1336-1340
Gough NM (1988) Rapid and quantitative preparation of cytoplasmic RNA from small
numbers of cells. Analytic Biochem. 173:93-95
Gruber A and Zingales B (1995) One-base double-stranded DNA sequencing using commercial kits. BioTechniques 19:748-750
Roopes BC, McClure WR (1981) Sturlies on the selectivity ofDNA precipitation by spermine. Nucleic Acids Res 9:5493-5504
Liedke W, Battistini L, Brosnan CF, Raine CS (1994} PCR Methods Applic 3:185-187
Okayama R, Berg P (1982) Righ-efficiency cloning of full-length cDNA. Mol Cell Biol
2:161-170
Sambrook J, Fritsch, EF, Maniatis T ( 1989} Molecular cloning. A laboratory manual. Cold
Spring Rarbor Laboratory Press, Cold Spring Rarbor
Shapiro DJ (1981) Quantitativeethanol precipitation of nanogram quantities of DNA
and RNA. Analytical Bioehern 110:229-231
Wallace DM (1987} Large- and small-scale phenol extraction. Methods Enzymol 152:
33-41