8. 1 M Dithiothreitol (DTT): Weigh 7.71 mg in 0.5 mL microcentrifuge tube and dissolve in 50 μL of autoclaved water (see
Note 2).
9. 5 μM Promoter-template DNA duplex: Transfer 5 μL of promoter DNA ON 11 (100 μM) and template DNA ON 12
(100 μM) into a 1.5 mL microcentrifuge tube containing
90 μL of the annealing buffer. Vortex the solution and centrifuge for 10 s. Anneal the ON mix by placing the centrifuge
tube on the heating block at 90
C for 3 min. Remove the
heating block from the dry-block heater and allow it to cool to
room temperature (RT). Place the annealed promotertemplate DNA duplex (final conc. 5 μM) in an ice bath for
30 min. Store the promoter-template duplex at À40
C.
2.2 PAGE Equipment
1. Gel electrophoresis equipment.
2. Glass plates (45 cm  20 cm, 4.8 mm thick).
3. Power supply.
4. 1.5 mm Spacer.
5. 1.5 mm Comb.
6. Plastic wrap (UV transparent).
7. Sterile scalpel.
8. Sterile glass rod.
9. Poly-Prep columns (Bio-Rad Laboratories).
10. Sep-Pak C18 cartridges (Waters Corporation).
11. UV lamp.
12. Rotary mixture.
2.3 PAGE Purification
of IU-Labeled RNA
Transcript
1. 10Â TBE resolving buffer: 0.89 M Tris, 0.89 M boric acid, and
20 mM EDTA. Dissolve 108 g Tris base and 55 g boric acid in
800 mL autoclaved water. To this solution add 40 mL of 0.5 M
EDTA (pH 8.0). Adjust the volume to 1 L with autoclaved
water. Filter the buffer through 0.45 μm filter paper. Store the
buffer at RT.
2. Denaturing acrylamide/bis-acrylamide solution (19:1): Weigh
190 g of acrylamide and 10 g of bis-acrylamide in 1 L reagent
bottle. Add autoclaved water to a volume of 300 mL. Further
add 420 g of urea followed by addition of 100 mL 10Â TBE
buffer. Stir the mixture vigorously using a magnetic stir bar.
Make up the volume to 1000 mL with autoclaved water and
filter using 0.45 μm filter paper. Store this solution at 4
C
protected from light (see Note 3).
3. 10% (wt/vol) Ammonium persulfate (APS): Weigh 1 g of APS
in 15 mL centrifuge tube and make the volume up to 10 mL by
adding autoclaved water (see Note 4).
Labeling RNA for Conformational Analysis and Imaging
477
Note 2).
9. 5 μM Promoter-template DNA duplex: Transfer 5 μL of promoter DNA ON 11 (100 μM) and template DNA ON 12
(100 μM) into a 1.5 mL microcentrifuge tube containing
90 μL of the annealing buffer. Vortex the solution and centrifuge for 10 s. Anneal the ON mix by placing the centrifuge
tube on the heating block at 90
C for 3 min. Remove the
heating block from the dry-block heater and allow it to cool to
room temperature (RT). Place the annealed promotertemplate DNA duplex (final conc. 5 μM) in an ice bath for
30 min. Store the promoter-template duplex at À40
C.
2.2 PAGE Equipment
1. Gel electrophoresis equipment.
2. Glass plates (45 cm  20 cm, 4.8 mm thick).
3. Power supply.
4. 1.5 mm Spacer.
5. 1.5 mm Comb.
6. Plastic wrap (UV transparent).
7. Sterile scalpel.
8. Sterile glass rod.
9. Poly-Prep columns (Bio-Rad Laboratories).
10. Sep-Pak C18 cartridges (Waters Corporation).
11. UV lamp.
12. Rotary mixture.
2.3 PAGE Purification
of IU-Labeled RNA
Transcript
1. 10Â TBE resolving buffer: 0.89 M Tris, 0.89 M boric acid, and
20 mM EDTA. Dissolve 108 g Tris base and 55 g boric acid in
800 mL autoclaved water. To this solution add 40 mL of 0.5 M
EDTA (pH 8.0). Adjust the volume to 1 L with autoclaved
water. Filter the buffer through 0.45 μm filter paper. Store the
buffer at RT.
2. Denaturing acrylamide/bis-acrylamide solution (19:1): Weigh
190 g of acrylamide and 10 g of bis-acrylamide in 1 L reagent
bottle. Add autoclaved water to a volume of 300 mL. Further
add 420 g of urea followed by addition of 100 mL 10Â TBE
buffer. Stir the mixture vigorously using a magnetic stir bar.
Make up the volume to 1000 mL with autoclaved water and
filter using 0.45 μm filter paper. Store this solution at 4
C
protected from light (see Note 3).
3. 10% (wt/vol) Ammonium persulfate (APS): Weigh 1 g of APS
in 15 mL centrifuge tube and make the volume up to 10 mL by
adding autoclaved water (see Note 4).
Labeling RNA for Conformational Analysis and Imaging
477