4. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED).
5. Denaturing loading buffer: 7 M Urea, 10 mM Tris–HCl,
100 mM EDTA, pH 8.0. Dissolve 4.2 g urea and 15.76 mg
Tris–HCl in 5 mL of autoclaved water. Add 2 mL of 0.5 M
EDTA (pH 8.0). Adjust the volume to 10 mL. Store the buffer
at room temperature.
6. 0.3 M Sodium acetate solution: Prepare 20 mL of 0.3 M
sodium acetate solution in autoclaved water.
2.4 Posttranscriptional
Suzuki-Miyaura
Coupling Reactions
1. 100 mM Stock solution of boronic acids/esters: Prepare 50 μL
solution of each substrate by dissolving respective amount of
boronic acid/ester substrates in BioUltra-grade DMSO (see
Note 5). Vortex the solution, spin, and store at À40
C (see
Note 5).
2. Pd-ligand catalyst (see Note 6): Add ADHP (65 mg, 0.5 mmol,
2 equiv.) or DMADHP (78 mg, 0.5 mmol, 2 equiv.) to a 5 mL
volumetric flask containing 3 mL of autoclaved water. Add
100 μL of 10 mM NaOH (4 mole equivalents) to above
solution. Stir the solution for 5 min at RT until all solids
dissolve completely. Add 56 mg of Pd(OAc) 2 (0.25 mmol,
1 mol equivalent) to the flask. Stir the mixture at 65
C for
60 min. Allow the solution to attain RT. Remove the stir bar
and adjust the volume to 5 mL with autoclaved water to make a
50 mM stock solution of the Pd-ligand catalyst. Store the
catalyst at À40
C. For cross-coupling reactions use 10 mM
solution of the catalyst diluted using autoclaved water. Store
10 mM stock solution at À40
C (see Note 6).
3. 250 mM Tris–HCl reaction buffer (5Â), pH 8.5: Weigh
303 mg of Tris base in 15 mL centrifuge tube. Dissolve Tris
base in 8 mL of autoclaved water. Adjust the pH of the solution
to 8.5 by adding 1 M HCl. Adjust the volume to 10 mL by
adding autoclaved water and filter the buffer using 0.22 μm
syringe filter. Store the buffer at room temperature.
2.5 HPLC Purification
of Cross-Coupled RNA
Products
1. 1 M Triethylammonium acetate buffer (TEAA), pH 7.0: Take
139.4 mL of HPLC-grade triethylamine in 1 L reagent bottle.
Add 700 mL of autoclaved water and stir the mixture on an ice
bath. Add dropwise 57.2 mL HPLC-grade acetic acid with
stirring. Adjust the pH of the solution to 7.0 and make the
total volume to 1 L by adding autoclaved water. Check the pH
again and store the buffer at 4
C. From this stock make 1 L of
50 mM solution of TEAA in autoclaved water. Filter the buffer
through 0.22 μm filter paper prior to HPLC purification.
2. Phenomenex-Luna C18 column (250 Â 4.6 mm, 5 μm) or an
equivalent column.
478
Manisha B. Walunj and Seergazhi G. Srivatsan
0 ,N
0 -tetramethylethylenediamine (TEMED).
5. Denaturing loading buffer: 7 M Urea, 10 mM Tris–HCl,
100 mM EDTA, pH 8.0. Dissolve 4.2 g urea and 15.76 mg
Tris–HCl in 5 mL of autoclaved water. Add 2 mL of 0.5 M
EDTA (pH 8.0). Adjust the volume to 10 mL. Store the buffer
at room temperature.
6. 0.3 M Sodium acetate solution: Prepare 20 mL of 0.3 M
sodium acetate solution in autoclaved water.
2.4 Posttranscriptional
Suzuki-Miyaura
Coupling Reactions
1. 100 mM Stock solution of boronic acids/esters: Prepare 50 μL
solution of each substrate by dissolving respective amount of
boronic acid/ester substrates in BioUltra-grade DMSO (see
Note 5). Vortex the solution, spin, and store at À40
C (see
Note 5).
2. Pd-ligand catalyst (see Note 6): Add ADHP (65 mg, 0.5 mmol,
2 equiv.) or DMADHP (78 mg, 0.5 mmol, 2 equiv.) to a 5 mL
volumetric flask containing 3 mL of autoclaved water. Add
100 μL of 10 mM NaOH (4 mole equivalents) to above
solution. Stir the solution for 5 min at RT until all solids
dissolve completely. Add 56 mg of Pd(OAc) 2 (0.25 mmol,
1 mol equivalent) to the flask. Stir the mixture at 65
C for
60 min. Allow the solution to attain RT. Remove the stir bar
and adjust the volume to 5 mL with autoclaved water to make a
50 mM stock solution of the Pd-ligand catalyst. Store the
catalyst at À40
C. For cross-coupling reactions use 10 mM
solution of the catalyst diluted using autoclaved water. Store
10 mM stock solution at À40
C (see Note 6).
3. 250 mM Tris–HCl reaction buffer (5Â), pH 8.5: Weigh
303 mg of Tris base in 15 mL centrifuge tube. Dissolve Tris
base in 8 mL of autoclaved water. Adjust the pH of the solution
to 8.5 by adding 1 M HCl. Adjust the volume to 10 mL by
adding autoclaved water and filter the buffer using 0.22 μm
syringe filter. Store the buffer at room temperature.
2.5 HPLC Purification
of Cross-Coupled RNA
Products
1. 1 M Triethylammonium acetate buffer (TEAA), pH 7.0: Take
139.4 mL of HPLC-grade triethylamine in 1 L reagent bottle.
Add 700 mL of autoclaved water and stir the mixture on an ice
bath. Add dropwise 57.2 mL HPLC-grade acetic acid with
stirring. Adjust the pH of the solution to 7.0 and make the
total volume to 1 L by adding autoclaved water. Check the pH
again and store the buffer at 4
C. From this stock make 1 L of
50 mM solution of TEAA in autoclaved water. Filter the buffer
through 0.22 μm filter paper prior to HPLC purification.
2. Phenomenex-Luna C18 column (250 Â 4.6 mm, 5 μm) or an
equivalent column.
478
Manisha B. Walunj and Seergazhi G. Srivatsan