3 Purification of Nucleic Acids From Eukaryotic Cells
63
Procedure
1. In advance prepare the following solutions:
Lysis Buffer
Component
a. Tris-HCl (pH 7.5)
b. NaCl
(DEPC-traeted)
c. MgCh
(DEPC-treated)
d. NP-40
e. HzO
(DEPC-treated)
Extraction buffer
Component
a. Tris-HCl (pH7.5)
b. NaCl (DEPC-treated)
c. EDTA (DEPC-treated)
d. SDS
e. urea
f. H20 (DEPC-treated)
Stock
Concentration
1M
SM
1M
100%
Stock
Concentration
1M
SM
0.5 M
10%
Final
Concentration
10mM
150mM
1.5 mM
0.65%
Final
Concentration
10mM
350mM
lOmM
1%
7M
Amount
0.5 ml
1.5 ml
0.075 ml
0.325 ml
46.925 ml
Amount
O.Sml
3.5 ml
1 ml
5 ml
21.02 g
to 50 ml
Note: For DEPC-treatment, see Subprotocol 3, Precautions. DEPC is suspected to be a carcinogen.
2. Transfer up to 5 x 10 6 cells directly from medium without washing in a
microcentrifuge tube.
3. Spin for 5min at 1000 rpm (without brake).
4. Remove supernatant.
5. Resupend pellet in 200 !J.l ice-cold lysis buffer, vortex rigorously, until
pellet is dissolved.
ln advance
Preparation of
cytoplasmic RNA