18. Hybridization oven.
19. Glass slides for microscopy.
20. Glass coverslips.
21. Clear nail polish.
2.2 Plant Growth
Material
1. Arabidopsis thaliana Col-0 seeds.
2. 6% (v/v) Sodium hypochlorite solution.
3. 0.1% (w/v) Agarose in water, freshly autoclaved.
4. Squared petri dishes.
5. ½ Murashige and Skoog (MS) plates: ½ MS medium including
vitamins and MES buffer pH 5.8 adjusted by potassium
hydroxide and 0.8% (w/v) plant agar, autoclaved and poured
into squared plates.
2.3 Synthesis
of Labeled RNA Probes
1. PCR kit.
2. RNase-free double-distilled water.
3. T7 RNA polymerase (store at À20
C).
4. SP6 RNA polymerase (store at À20
C).
5. 10Â Transcription buffer (supplied with the RNA polymerase).
6. 10Â DIG RNA-labeling mix: 10 mM ATP, 10 mM CTP,
10 mM GTP, 6.5 mM UTP, 3.5 mM digoxigenin-11-UTP
(store at À20
C).
7. 10Â Fluorescein RNA-labeling mix: 10 mM ATP, 10 mM
CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM fluorescein-12UTP, pH 7.5 (store at À20
C).
8. RNase inhibitor (store at À20
C).
9. 20 mg/mL tRNA from E. coli (in RNase-free water; store in
aliquots at À20
C).
10. DNase I (RNase-free; store at À20
C).
11. 0.5 M Ethylene-diamine-tetra-acetic acid (EDTA), pH 8.0
(RNase free).
12. 10 M Lithium chloride (LiCl), diethyl pyrocarbonate (DEPC)
treated (see Subheading 3.1, step 4).
13. 2Â Alkaline hydrolysis buffer: Mix freshly 600 μL of RNasefree double-distilled water, 240 μL of 0.5 M Na 2 CO 3 , and
160 μL of 0.5 M NaHCO 3 .
14. Acetic acid.
15. 3 M Sodium acetate.
16. 10Â 3-Morpholinopropane-1-sulfonic acid (MOPS) buffer:
0.4 M MOPS, 0.1 M sodium acetate, 0.01 M EDTA,
pH 7.0. Prepare in RNase-free double-distilled water and
RNA-FISH
7