68
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
• methanol
• DEPC-treated water
Supplies
• oligo(dT)-cellulose (0.5-1 ml packed volume)
• glass wool and pasteur pipet or sterile small disposable column with a
2 ml capacity
• sterilized microcentrifuge tubes
• microcentrifuge at 4°C
• spectrophotometer
Procedure
ln advance
1. In advance prepare the following solutions:
- 2 x column-loading buffera
Component
Stock
Final
Concentration
Concentration
Tris-HCl (pH 7.5)
1M
40mM
LiCl (DEPC-treated)
SM
IM
EDTA (DEPC-treated)
0.5 M
2mM
sodium lauryl sarcosinateb
10%
0.2%
H20 (DEPC-treated)
Amount
4 ml
20 ml
0.4 ml
2 ml
73.6 ml
• Do not sterilize solution by autoclaving, since it will froth extensively.
b Heat up sodium lauryl sarcosinate (10%) to 65°C for 30 min.
- Elution buffera
Component
Stock
Final
Concentration
Concentration
Tris-HCl (pH7.5)
1M
lOmM
EDTA (DEPC-treated)
O.SM
O.lmM
DS
10%
0.05%
H20 (DEPC-treated)
• Do not sterilize solution by autoclaving, since it will froth.
Amount
O.Sml
O.lml
0.25ml
48.25ml