26. 10Â TBE: 900 mM Tris, 20 mM EDTA, 900 mM boric acid.
Weigh 109 g of Tris and 55.6 g of boric acid and transfer to a
glass beaker. Add 40 mL of 0.5 M EDTA, pH 8.0, and water to
a volume of about 900 mL, and mix. Transfer to a glass
cylinder and adjust volume to 1 L with water. Autoclave and
store at room temperature (see Note 3).
27. 1Â TBE: 90 mM Tris, 2 mM EDTA, 90 mM boric acid. Mix
100 mL of 10Â TBE with 900 mL of water in a glass cylinder.
Mix well and store at room temperature.
28. 0.5Â TBE: 45 mM Tris, 1 mM EDTA, 45 mM boric acid. Mix
50 mL of 10Â TBE with 950 mL of water in a glass cylinder.
Mix well and store at room temperature.
29. 15% Denaturing acrylamide gel: 15% Acrylamide/bisacrylamide 19/1, 7 M urea, 1Â TBE. Weigh 84 g of urea and
transfer to a glass beaker. Add 75 mL of 40% acrylamide/bisacrylamide (19:1) and 20 mL of 10Â TBE. Add water to a
volume of 190 mL and mix thoroughly until the urea is
completely dissolved. Transfer to a cylinder and adjust volume
to 200 mL with water. Mix well and store at room temperature
in a bottle wrapped in aluminum foil to avoid light exposure.
30. 10% (w/v) Ammonium persulfate (APS): Store at 4
C.
31. N,N,N,N
0 -tetramethylethylenediamine (TEMED): Store at
4
C.
32. 1% Ethidium bromide: Store at 4
C.
33. G-50 resin: Pour about 1 mL of Sephadex G-50 resin powder
in a 14 mL round-bottom tube. Add about 10 mL of water and
mix well. Autoclave and store at 4
C.
34. PerfectHyb™ Plus hybridization buffer (Sigma-Aldrich).
35. 20Â Saline sodium citrate (SSC): 3 M NaCl, 300 mM trisodium citrate, pH 7.0. Weigh 175.3 g of NaCl and 88.2 g of
sodium citrate dihydrate and transfer to a glass beaker. Add
about 500 mL of water, and mix. Adjust pH to 7.0 with 99%
pure acetic acid. Transfer to a glass cylinder and adjust volume
to 1 L with water. Autoclave and store at room temperature.
36. 2Â SSC: 0.3 M NaCl, 30 mM trisodium citrate, pH 7.0. Mix
50 mL of 20Â SSC with 450 mL of water in a glass cylinder.
Mix well and store at room temperature.
37. 10% (w/v) Sodium dodecyl sulfate (SDS) (see Note 4): Autoclave and store at room temperature.
38. 2Â SSC, 0.1% SDS: Transfer 50 mL of 20Â SSC into a 500 mL
cylinder. Add 400 mL of water and then 5 mL of 10% SDS.
Adjust volume to 500 mL by gently adding water to avoid
foaming. Mix and store at room temperature (see Note 5).
Plant Protoplasts-Based System to Transfect RNA
417
Weigh 109 g of Tris and 55.6 g of boric acid and transfer to a
glass beaker. Add 40 mL of 0.5 M EDTA, pH 8.0, and water to
a volume of about 900 mL, and mix. Transfer to a glass
cylinder and adjust volume to 1 L with water. Autoclave and
store at room temperature (see Note 3).
27. 1Â TBE: 90 mM Tris, 2 mM EDTA, 90 mM boric acid. Mix
100 mL of 10Â TBE with 900 mL of water in a glass cylinder.
Mix well and store at room temperature.
28. 0.5Â TBE: 45 mM Tris, 1 mM EDTA, 45 mM boric acid. Mix
50 mL of 10Â TBE with 950 mL of water in a glass cylinder.
Mix well and store at room temperature.
29. 15% Denaturing acrylamide gel: 15% Acrylamide/bisacrylamide 19/1, 7 M urea, 1Â TBE. Weigh 84 g of urea and
transfer to a glass beaker. Add 75 mL of 40% acrylamide/bisacrylamide (19:1) and 20 mL of 10Â TBE. Add water to a
volume of 190 mL and mix thoroughly until the urea is
completely dissolved. Transfer to a cylinder and adjust volume
to 200 mL with water. Mix well and store at room temperature
in a bottle wrapped in aluminum foil to avoid light exposure.
30. 10% (w/v) Ammonium persulfate (APS): Store at 4
C.
31. N,N,N,N
0 -tetramethylethylenediamine (TEMED): Store at
4
C.
32. 1% Ethidium bromide: Store at 4
C.
33. G-50 resin: Pour about 1 mL of Sephadex G-50 resin powder
in a 14 mL round-bottom tube. Add about 10 mL of water and
mix well. Autoclave and store at 4
C.
34. PerfectHyb™ Plus hybridization buffer (Sigma-Aldrich).
35. 20Â Saline sodium citrate (SSC): 3 M NaCl, 300 mM trisodium citrate, pH 7.0. Weigh 175.3 g of NaCl and 88.2 g of
sodium citrate dihydrate and transfer to a glass beaker. Add
about 500 mL of water, and mix. Adjust pH to 7.0 with 99%
pure acetic acid. Transfer to a glass cylinder and adjust volume
to 1 L with water. Autoclave and store at room temperature.
36. 2Â SSC: 0.3 M NaCl, 30 mM trisodium citrate, pH 7.0. Mix
50 mL of 20Â SSC with 450 mL of water in a glass cylinder.
Mix well and store at room temperature.
37. 10% (w/v) Sodium dodecyl sulfate (SDS) (see Note 4): Autoclave and store at room temperature.
38. 2Â SSC, 0.1% SDS: Transfer 50 mL of 20Â SSC into a 500 mL
cylinder. Add 400 mL of water and then 5 mL of 10% SDS.
Adjust volume to 500 mL by gently adding water to avoid
foaming. Mix and store at room temperature (see Note 5).
Plant Protoplasts-Based System to Transfect RNA
417