62
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
• Several RNAse inhibitors are available, but they are expensive and are
mostly used at the end of the purification process.
Rapid preparation of cytoplasmic RNA by urea/SDS/phenol/chloroform/extraction
This protocols makes use of cellular fractionation by differential centrifugation. Genomic DNA is removed by pelleting the cell nuclei. The cytoplasmatic lysate is then urea/SDS/phenol/chloroform extracted. The procedure
is extremely rapid (30 min) and useful for processing of large numbers of
samples (Gough 1988).
Materials
Reagents • 5 x 10 6 cells
• phenol:chloroform:isoamyl alcohol (25:24:1)
• 1 M MgCh (DEPC-treated)
• 5 M NaCl (DEPC-treated)
• NP-40
• 10% SDS
• 1 M tris-HCl (pH 7.5)
• urea
• 70% and 100% ethanol, ice-cold
• RNAse-free TE-buffer (pH 7.0) (10 mM Tris-HCl (pH 7.0), 1 mM EDTA
(pH 8.0))
• DEPC-treated water
Supplies
• 1.5ml sterilized polypropylene tubes
• microcentrifuge at 4°C
• chemical hood
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
• Several RNAse inhibitors are available, but they are expensive and are
mostly used at the end of the purification process.
Rapid preparation of cytoplasmic RNA by urea/SDS/phenol/chloroform/extraction
This protocols makes use of cellular fractionation by differential centrifugation. Genomic DNA is removed by pelleting the cell nuclei. The cytoplasmatic lysate is then urea/SDS/phenol/chloroform extracted. The procedure
is extremely rapid (30 min) and useful for processing of large numbers of
samples (Gough 1988).
Materials
Reagents • 5 x 10 6 cells
• phenol:chloroform:isoamyl alcohol (25:24:1)
• 1 M MgCh (DEPC-treated)
• 5 M NaCl (DEPC-treated)
• NP-40
• 10% SDS
• 1 M tris-HCl (pH 7.5)
• urea
• 70% and 100% ethanol, ice-cold
• RNAse-free TE-buffer (pH 7.0) (10 mM Tris-HCl (pH 7.0), 1 mM EDTA
(pH 8.0))
• DEPC-treated water
Supplies
• 1.5ml sterilized polypropylene tubes
• microcentrifuge at 4°C
• chemical hood