5. Mix the above reaction mixture well with pipette and add
2.5 μL of 40 U/μL RiboLock.
6. Initiate the transcription reaction by adding 40 μL T7 RNA
polymerase (20 U/μL). Mix the reaction mixture by pipetting
up and down few times.
7. Incubate the reaction mixture at 37
C for 6 h (see Note 7).
3.2 PreparativeScale PAGE
Purification
of IU-Labeled RNA
Transcript
1. Mix 150 mL of 20% denaturing acrylamide/bis-acrylamide
solution with 1.4 mL of 10% aqueous APS in a 250 mL Erlenmeyer flask and add 70 μL of TEMED.
2. Pour the solution between the glass plates and insert a comb
(1.5 mm thick with 4 wells). Polymerize for ~1 h.
3. Rinse the wells thoroughly with Millipore or autoclaved water
to remove unpolymerized acrylamide and place it on the electrophoresis apparatus.
4. Fill the upper and lower chambers with 1Â TBE buffer;
pre-run the gel for at least 60 min at 25 W.
5. In the meantime, reduce the reaction volume (see Subheading
3.1) to nearly 1/3 in a speed vac (e.g., Labconco). Add 50 μL
denaturing loading buffer (see Note 8).
6. After pre-run, turn off the power supply and wash the wells
again thoroughly with 1Â TBE buffer.
7. Load the samples on the preparative 20% denaturing polyacrylamide gel using pipette.
8. Run the gel at constant 25 W for ~6 h.
9. Turn off the power supply and carefully separate the gel from
the plates. Cover the gel using plastic wrap.
10. Mark the band corresponding to the full-length transcript by
UV shadowing (254 nm). This is usually a slower migrating
intense band. Cut this band using a sterile scalpel.
11. Carefully transfer the gel pieces to a Poly-Prep column
(BioRad) and crush the gel with a sterile glass rod into fine
pieces.
12. Add 3 mL of 0.3 M aqueous sodium acetate solution and cap
the Poly-Prep column.
13. Place the Poly-Prep column on a rotary mixer and allow the
contents to mix well for ~12 h at RT.
14. Centrifuge the Poly-Prep column for 10–15 min and collect
the solution in a 15 mL centrifuge tube.
15. Meanwhile condition the reversed-phase Sep-Pak column
(Waters) by washing with 5 mL of acetonitrile and 15 mL of
autoclaved water. The Sep-Pak column can be easily connected
to a plastic syringe, which can be used for washing and loading
the sample.
480
Manisha B. Walunj and Seergazhi G. Srivatsan
2.5 μL of 40 U/μL RiboLock.
6. Initiate the transcription reaction by adding 40 μL T7 RNA
polymerase (20 U/μL). Mix the reaction mixture by pipetting
up and down few times.
7. Incubate the reaction mixture at 37
C for 6 h (see Note 7).
3.2 PreparativeScale PAGE
Purification
of IU-Labeled RNA
Transcript
1. Mix 150 mL of 20% denaturing acrylamide/bis-acrylamide
solution with 1.4 mL of 10% aqueous APS in a 250 mL Erlenmeyer flask and add 70 μL of TEMED.
2. Pour the solution between the glass plates and insert a comb
(1.5 mm thick with 4 wells). Polymerize for ~1 h.
3. Rinse the wells thoroughly with Millipore or autoclaved water
to remove unpolymerized acrylamide and place it on the electrophoresis apparatus.
4. Fill the upper and lower chambers with 1Â TBE buffer;
pre-run the gel for at least 60 min at 25 W.
5. In the meantime, reduce the reaction volume (see Subheading
3.1) to nearly 1/3 in a speed vac (e.g., Labconco). Add 50 μL
denaturing loading buffer (see Note 8).
6. After pre-run, turn off the power supply and wash the wells
again thoroughly with 1Â TBE buffer.
7. Load the samples on the preparative 20% denaturing polyacrylamide gel using pipette.
8. Run the gel at constant 25 W for ~6 h.
9. Turn off the power supply and carefully separate the gel from
the plates. Cover the gel using plastic wrap.
10. Mark the band corresponding to the full-length transcript by
UV shadowing (254 nm). This is usually a slower migrating
intense band. Cut this band using a sterile scalpel.
11. Carefully transfer the gel pieces to a Poly-Prep column
(BioRad) and crush the gel with a sterile glass rod into fine
pieces.
12. Add 3 mL of 0.3 M aqueous sodium acetate solution and cap
the Poly-Prep column.
13. Place the Poly-Prep column on a rotary mixer and allow the
contents to mix well for ~12 h at RT.
14. Centrifuge the Poly-Prep column for 10–15 min and collect
the solution in a 15 mL centrifuge tube.
15. Meanwhile condition the reversed-phase Sep-Pak column
(Waters) by washing with 5 mL of acetonitrile and 15 mL of
autoclaved water. The Sep-Pak column can be easily connected
to a plastic syringe, which can be used for washing and loading
the sample.
480
Manisha B. Walunj and Seergazhi G. Srivatsan