54
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
6. Repeat steps 2-5, as long as white flocculent interface is present.
7. Add an equal volume of chloroform and repeat steps 3-5.
8. Add 0.1 vol of 3M sodium acetate (pH 5.2). Mix by vortexing briefly or
by flicking the tube several tim es. Add 2.5 vol of ice-cold 100% ethanol.
9. Keep at 4 oc for 10 min.
10. Spinat 15000 rpm for 10 min.
11. Remove the ethanol carefully.
12. Add 1 ml 70% ethanol to remove precipitated salt, vortex briefly.
13. Repeat steps 10 and 11.
14. Dry pellet in a desiccator under vacuum or in a Speedvac® evaporator.
Note: Genomic DNA and large DNA fragments should not be dried completely because rehydration may take days. Air dry genomic DNA under
sight and dissolve pellet when still moist and shiny.
15. Resuspend pellet in TE-buffer at < 1mg/ml and store at -20 oc or at 4 oc,
when used frequently. RNA is stored at -70 oc under 70-80% ethanol.
Note: High-molecular-weight genomic DNA may require one to several
days to dissolve and should be shaken gently to avoid shearing.
16. Analyse the quantity and quality of the nucleic acid content by spectrophotometric determination of an aliquot at wave-lengths of 260 nm and
280 nm.
Note: The ratio between the readings at 260 nm and 280 nm provides an
estimate of the purity of the nucleic acids and should be between 1.8
and 2.0. Cantamination by phenol or proteinwill result in ratios significantly less than 1.8. An OD of 1 at 260 nm corresponds to approximately
50 f.lg/ml for double-stranded DNA, 40 flg/ml for single-stranded DNA and
RNA and -20 f.lg/ml for single-stranded oligonucleotides.
Comments
Optimizing extraction conditions
• Technical considerations.
Of fundamental importance for successful phenol extraction of intact
nucleic acids is the performance of all steps in one session ( especially
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
6. Repeat steps 2-5, as long as white flocculent interface is present.
7. Add an equal volume of chloroform and repeat steps 3-5.
8. Add 0.1 vol of 3M sodium acetate (pH 5.2). Mix by vortexing briefly or
by flicking the tube several tim es. Add 2.5 vol of ice-cold 100% ethanol.
9. Keep at 4 oc for 10 min.
10. Spinat 15000 rpm for 10 min.
11. Remove the ethanol carefully.
12. Add 1 ml 70% ethanol to remove precipitated salt, vortex briefly.
13. Repeat steps 10 and 11.
14. Dry pellet in a desiccator under vacuum or in a Speedvac® evaporator.
Note: Genomic DNA and large DNA fragments should not be dried completely because rehydration may take days. Air dry genomic DNA under
sight and dissolve pellet when still moist and shiny.
15. Resuspend pellet in TE-buffer at < 1mg/ml and store at -20 oc or at 4 oc,
when used frequently. RNA is stored at -70 oc under 70-80% ethanol.
Note: High-molecular-weight genomic DNA may require one to several
days to dissolve and should be shaken gently to avoid shearing.
16. Analyse the quantity and quality of the nucleic acid content by spectrophotometric determination of an aliquot at wave-lengths of 260 nm and
280 nm.
Note: The ratio between the readings at 260 nm and 280 nm provides an
estimate of the purity of the nucleic acids and should be between 1.8
and 2.0. Cantamination by phenol or proteinwill result in ratios significantly less than 1.8. An OD of 1 at 260 nm corresponds to approximately
50 f.lg/ml for double-stranded DNA, 40 flg/ml for single-stranded DNA and
RNA and -20 f.lg/ml for single-stranded oligonucleotides.
Comments
Optimizing extraction conditions
• Technical considerations.
Of fundamental importance for successful phenol extraction of intact
nucleic acids is the performance of all steps in one session ( especially