64
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
6. Spin for 5 min at 1000 rpm, 4°C, to pellet nuclei.
7. Transfer supernatant (cytoplasmatic lysate) to a fresh microcentrifuge
tube containing 200 J..ll ice-cold extraction buffer and 400 J..ll phenol:chloroform:isoamyl alcohol (25:24:1), vortex rigorously.
Note: Sampies may be stored at this stage at -70°C for up to several weeks.
8. Spin for 2 min at 15000 rpm.
9. Transfer top (aqueous) phase to a new microcentrifuge tube.
10. Add 1 ml ice-cold 100% ethanol and chill for 20 min at -20°C.
11. Spin at 15000 rpm for 15 min.
12. Remove the ethanol carefully.
13. Add 1 ml 70% ethanol to remove precipitated salt, vortex briefly.
14. Repeat steps 11 and 12.
15. Dry pellet.
16. Resuspend RNA pellet in 200 J..ll TE-buffer.
17. Mix 10 J..ll of the RNA sample and 490 J..ll buffer and measure the absorbance of the solution at 260 nm.
18. If not using the remaining RNA sample immediately, add 500 J..ll icecold 100% ethanol and store at -70°C. RNA can be applied directly
to formaldehyde-agarose gels or RT-PCR.
Subprotocol 4
Rapid Preparation of Total RNA by Acid Guanidinium Thiocyanate/
Phenoi/Chloroform/Extraction
For principles and precautions see Subprotocol 3, Introduction.
Guanidinium thiocyanate is one of the most effective protein denaturants and facilitates the isolation ofintact, functional RNA from a wide range
ofbiological sources, even those with extraordinarily high Ievels of nuclease
like pancreatic cells. Denaturation is enhanced by including ß-mercaptoethanol, a reductant, which breaks intramolecular protein disulfide
bonds. This method, established by Chomczynski and Sacchi (1987), provides both high and pure yields of undegraded RNA and can easily be scaled
up or down.
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
6. Spin for 5 min at 1000 rpm, 4°C, to pellet nuclei.
7. Transfer supernatant (cytoplasmatic lysate) to a fresh microcentrifuge
tube containing 200 J..ll ice-cold extraction buffer and 400 J..ll phenol:chloroform:isoamyl alcohol (25:24:1), vortex rigorously.
Note: Sampies may be stored at this stage at -70°C for up to several weeks.
8. Spin for 2 min at 15000 rpm.
9. Transfer top (aqueous) phase to a new microcentrifuge tube.
10. Add 1 ml ice-cold 100% ethanol and chill for 20 min at -20°C.
11. Spin at 15000 rpm for 15 min.
12. Remove the ethanol carefully.
13. Add 1 ml 70% ethanol to remove precipitated salt, vortex briefly.
14. Repeat steps 11 and 12.
15. Dry pellet.
16. Resuspend RNA pellet in 200 J..ll TE-buffer.
17. Mix 10 J..ll of the RNA sample and 490 J..ll buffer and measure the absorbance of the solution at 260 nm.
18. If not using the remaining RNA sample immediately, add 500 J..ll icecold 100% ethanol and store at -70°C. RNA can be applied directly
to formaldehyde-agarose gels or RT-PCR.
Subprotocol 4
Rapid Preparation of Total RNA by Acid Guanidinium Thiocyanate/
Phenoi/Chloroform/Extraction
For principles and precautions see Subprotocol 3, Introduction.
Guanidinium thiocyanate is one of the most effective protein denaturants and facilitates the isolation ofintact, functional RNA from a wide range
ofbiological sources, even those with extraordinarily high Ievels of nuclease
like pancreatic cells. Denaturation is enhanced by including ß-mercaptoethanol, a reductant, which breaks intramolecular protein disulfide
bonds. This method, established by Chomczynski and Sacchi (1987), provides both high and pure yields of undegraded RNA and can easily be scaled
up or down.