224
HIDEYO OHUCHI AND SUMIHARE NOJI
Labware - 24-well disposable dish
Reagents - Enzymes (Boehringer, Promega etc.)
- RNA polymerase (appropriate polymerase; T7, T3, SP6)
- RNase Inhibitor
- DNase (RNase-free)
- Nucleotide mix (pH7.S; or DIG RNA Labeling Mix, lOx cone;
Boehringer)
- lOmM GTP
- lOmM ATP
- IOmM CTP
- 6.5mM UTP
- 3.5mM digoxigenin-UTP
- Sx transcription buffer (or use buffer supplied by supplier of
RNA polymerase)
- 200mM Tris HCI (pH7.9)
- 30mM MgCh
- IOmM spermidine
- SOmM NaCI
- Reagents for agarose gel electrophoresis (RNase-free):
- Agarose
- Ix TAE or Ix TBE
- IOmg/ml Ethidium bromide
- DNA or RNA marker
- 4M LiCI
- lOmg/ml yeast tRNA
Yeast tRNA powder (molecular biology grade) is available
from GIBCO-BRL. Dissolve the powder in sterile dH20 and
store in aliquats at -20°e.
- Glacial acetic acid
- Ethanol (200-proof)
- dH20 (RNase-free)
- Buffers for alkaline hydrolysis (store in aliquots at -20°C)
- 80mM NaHC03
- 120mM Na2C03
- PBS (phosphate buffered saline; autoclave and store at 4°C)
- lOmM Sodium Phosphate (pH 7.4)
- lOOmM NaCI
- Autoclave.
- O.2M Sodium phosphate; solution A: solution B= 22.6: 77.4