2.12 RNA Isolation
1. Proteinase K buffer: 100 mM Tris–HCl pH 7.4, 50 mM NaCl,
10 mM EDTA.
2. Proteinase K (Roche): 20 mg/mL in 10 mM Tris–HCl,
pH 7.4.
3. Proteinase K urea buffer: 100 mM Tris–HCl, pH 7.4, 50 mM
NaCl, 10 mM EDTA, 7 M urea.
4. Tri-reagent: 0.8 M Guanidinium thiocyanate, 0.4 M ammonium thiocyanate, 4.35% (w/v) glycerol, 0.1 M sodium acetate, pH 5.0, 38% (v/v) acidic phenol.
5. Chloroform:isoamyl alcohol (24:1).
6. Isopropanol.
7. 70% (v/v) Ethanol pre-chilled at À20
C.
8. Ambion
® GlycoBlue™ (Invitrogen).
9. 3 M Sodium acetate, pH 5.5.
10. 100% Ethanol, pre-chilled.
11. Thermomixer.
12. Phase Lock Gel heavy tubes (VWR).
3 Methods
3.1 Plant Growth
1. Grow Arabidopsis plants of the appropriate genotypes for
2–3 weeks on agar plates under the chosen experimental
conditions.
3.2 UV Light Fixation
and Plant Harvest
1. Place agar plates on ice inside the UV cross-linker.
2. Subject plants to cross-linking, starting with a dose of
500 mJ/cm
2 (see Note 4).
3. Harvest plants and immediately transfer to liquid nitrogen.
4. Grind frozen plant material to a fine powder in liquid nitrogen.
5. Transfer 0.5 g ground plant material to pre-chilled 2 mL tubes
with punctured lid. Use 2 Â 0.5 g distributed to two 2 mL
tubes. Store at À80
C until further use.
3.3 Preparation
of Beads
1. To prepare sepharose beads for preclearing (see Note 5),
pipette 100 μl of sepharose beads (50% slurry) (see Note 6) in
a 1.5 mL tube and add 1 mL of ice-cold IP lysis buffer w/o
inhibitors. Incubate for 5 min at 4
C in an end-over-end
rotator. Centrifuge for 1 min at 500 Â g. Repeat this washing
step in IP lysis buffer twice. Store at 4
C until use.
2. To prepare GFP-Trap
® beads for immunoprecipitation, wash
20 μL GFP-Trap
® beads (50% slurry) with 1 mL of ice-cold
IP lysis buffer in a 1.5 mL LoBind
® tube for 5 min at 4
C
260
Tino Ko ¨ ster and Dorothee Staiger
1. Proteinase K buffer: 100 mM Tris–HCl pH 7.4, 50 mM NaCl,
10 mM EDTA.
2. Proteinase K (Roche): 20 mg/mL in 10 mM Tris–HCl,
pH 7.4.
3. Proteinase K urea buffer: 100 mM Tris–HCl, pH 7.4, 50 mM
NaCl, 10 mM EDTA, 7 M urea.
4. Tri-reagent: 0.8 M Guanidinium thiocyanate, 0.4 M ammonium thiocyanate, 4.35% (w/v) glycerol, 0.1 M sodium acetate, pH 5.0, 38% (v/v) acidic phenol.
5. Chloroform:isoamyl alcohol (24:1).
6. Isopropanol.
7. 70% (v/v) Ethanol pre-chilled at À20
C.
8. Ambion
® GlycoBlue™ (Invitrogen).
9. 3 M Sodium acetate, pH 5.5.
10. 100% Ethanol, pre-chilled.
11. Thermomixer.
12. Phase Lock Gel heavy tubes (VWR).
3 Methods
3.1 Plant Growth
1. Grow Arabidopsis plants of the appropriate genotypes for
2–3 weeks on agar plates under the chosen experimental
conditions.
3.2 UV Light Fixation
and Plant Harvest
1. Place agar plates on ice inside the UV cross-linker.
2. Subject plants to cross-linking, starting with a dose of
500 mJ/cm
2 (see Note 4).
3. Harvest plants and immediately transfer to liquid nitrogen.
4. Grind frozen plant material to a fine powder in liquid nitrogen.
5. Transfer 0.5 g ground plant material to pre-chilled 2 mL tubes
with punctured lid. Use 2 Â 0.5 g distributed to two 2 mL
tubes. Store at À80
C until further use.
3.3 Preparation
of Beads
1. To prepare sepharose beads for preclearing (see Note 5),
pipette 100 μl of sepharose beads (50% slurry) (see Note 6) in
a 1.5 mL tube and add 1 mL of ice-cold IP lysis buffer w/o
inhibitors. Incubate for 5 min at 4
C in an end-over-end
rotator. Centrifuge for 1 min at 500 Â g. Repeat this washing
step in IP lysis buffer twice. Store at 4
C until use.
2. To prepare GFP-Trap
® beads for immunoprecipitation, wash
20 μL GFP-Trap
® beads (50% slurry) with 1 mL of ice-cold
IP lysis buffer in a 1.5 mL LoBind
® tube for 5 min at 4
C
260
Tino Ko ¨ ster and Dorothee Staiger