120
HIDEFUMI YOSHIOKA AND SUMIHARE NOTI
Preparation of
RI-Iabeled
riboprobes
Incorporation
ratio
(optional)
3. Place in a conventional paraffin microtome.
4. Cut the paraffin block into 5 11m serial sections.
5. Transfer sections to a drop of sterilized water on an APES
coated slide.
6. Dry slides on a warm plate at 42° C.
7. Store the slides at 4° C.
1. Make water bath at 37° C for T7 and T3 or at 40° C for SP6
RNA polymerase
2. Mixthese reagents inthe following order at room temperature.
For 5 tubes:
RNA polymerase buffer
20 ).11
10 mM rATP
5 ).11
10 mM rCTP
5 ).11
10 mM rGTP
5 ).11
10 mM rUTP
3.25 ).11
[35-Sj-rUTP, 14.8 -29.6 TBq (400-800 Ci Immol) 50 ).11
Sterile water
43.25 ).11
3. Transfer 17 III mixture to a 1.5 ml tube
4. Add 2 III linearized DNA to each tube.
5. Add 1 III RNase inhibitor
6. Add 1 III RNA polymerase.
7. Incubate for 2 hrs at 37° C for T7 or T3 RNA polymerase or at
40°C for SP6 RNA polymerase.
8. To check synthesis of RNA probes, remove a 1111 aliquot and
run on an agarose gel which does not need to be denatured.
An RNA band should belO-fold more intense than the plasmid band.
a) To check incorporation of RI in probes, remove 1111 reaction mixture to 499 III water.