122
PETER IGARASHI
tration of 0.2-1 mg/ml. V erify completeness of the digestion by agarose gel
electrophoresis.
Other reagents • 5x transcription buffer: 200 mM Tris-HCl (pH 8.0), 40 mM MgC}z, 250
Supplies and
equipment
mM N aCl, 10 mM spermidine
• Gelloading buffer (optional): 95% formamide, 20 mM EDTA, 0.05%
bromphenol blue, 0.05% xylene cyanol
• Gel elution buffer ( optional): 2 M ammonium acetate, 1 o/o SDS, 25 11g/ml
yeast tRNA
• Ribonuclease-free deoxyribonuclease I (RNase-free DNase, 1-10 unit/Jll)
• 10 mM each of rATP, rGTP, and rCTP; 1 mM UTP
• RNAse Block II (1 unit/Jll, Stratagene) or RNAsin (40 units/Jll, Promega)
• a[ 32 P]UTP {800 Ci/mmol, 10 mCi/ml) or a- 35 S-UTP (1000 Ci/mmol, 10
mCi/ml)
• Bacteriophage T3, T7 or SP6 RNA polymerase ( depending on the promoter present in the plasmid) ( diluted to 10 units/Jll in 1x transcription
buffer)
• Tris-equilibrated phenol
• Chloroform:isoamyl alcohol (24:1, v/v)
• 7.5 M ammonium acetate
• Absolute ethanol (stored at -20°C)
• DEPC-treated water (add 0.1 o/o diethylpyrocarbonate, then autoclave
prior to use)
• 37°C water bath
• Microcentrifuge
• Dry ice
• Sequencing gel electrophoresis apparatus and power supply ( optional)
• X-ray film (X-Ornat AR, Hyperfilm ß, or equivalent) (optional)
• Liquid scintillation counter
• Speed-Vac evaporator/concentrator
• Geiger counter
PETER IGARASHI
tration of 0.2-1 mg/ml. V erify completeness of the digestion by agarose gel
electrophoresis.
Other reagents • 5x transcription buffer: 200 mM Tris-HCl (pH 8.0), 40 mM MgC}z, 250
Supplies and
equipment
mM N aCl, 10 mM spermidine
• Gelloading buffer (optional): 95% formamide, 20 mM EDTA, 0.05%
bromphenol blue, 0.05% xylene cyanol
• Gel elution buffer ( optional): 2 M ammonium acetate, 1 o/o SDS, 25 11g/ml
yeast tRNA
• Ribonuclease-free deoxyribonuclease I (RNase-free DNase, 1-10 unit/Jll)
• 10 mM each of rATP, rGTP, and rCTP; 1 mM UTP
• RNAse Block II (1 unit/Jll, Stratagene) or RNAsin (40 units/Jll, Promega)
• a[ 32 P]UTP {800 Ci/mmol, 10 mCi/ml) or a- 35 S-UTP (1000 Ci/mmol, 10
mCi/ml)
• Bacteriophage T3, T7 or SP6 RNA polymerase ( depending on the promoter present in the plasmid) ( diluted to 10 units/Jll in 1x transcription
buffer)
• Tris-equilibrated phenol
• Chloroform:isoamyl alcohol (24:1, v/v)
• 7.5 M ammonium acetate
• Absolute ethanol (stored at -20°C)
• DEPC-treated water (add 0.1 o/o diethylpyrocarbonate, then autoclave
prior to use)
• 37°C water bath
• Microcentrifuge
• Dry ice
• Sequencing gel electrophoresis apparatus and power supply ( optional)
• X-ray film (X-Ornat AR, Hyperfilm ß, or equivalent) (optional)
• Liquid scintillation counter
• Speed-Vac evaporator/concentrator
• Geiger counter