total volume as the RNA sample aliquots, and then treat it like
the other samples for all remaining steps.
3. Freeze the samples at À20
C (overnight).
4. Dry the frozen samples completely by sublimation in a SpeedVac concentrator (45 min for a 20––30 μL sample volume
at RT).
5. Resuspend the samples in 12 μL RNA-loading buffer (see
Note 13).
6. Heat the samples to 95
C for 3 min, and then keep them
on ice.
7. After this RNA heat denaturation, add DynaMarker Prestain
“small RNA Plus” marker (5.5 μL) into the RNA oligo size
standard mix.
8. Set up the vertical gel apparatus for casting. Make sure that all
the components are clean.
9. Prepare a 16% polyacrylamide gel mixture in a 50 mL Falcon
tube, containing 11.1 g urea, 6.0 mL 5Â TBE, and 12.0 mL
acrylamide/bis-acrylamide (19:1). Fill with Milli-Q water up
to 30 mL and mix to dissolve all the components.
10. Rapidly add 30 μL TEMED and 300 μL 10% APS to the
mixture just before casting. Immediately cap and invert the
Falcon tube to mix. Then, without hesitation, steadily transfer
this solution between the glass plates of the gel apparatus using
a 10 mL pipette.
11. Add the comb while avoiding any air bubbles before the gel
starts to polymerize.
12. Let the gel sit for about 20–30 min for complete polymerization. Leftover gel mixture in the Falcon tube can be observed
as a reference for the polymerization reaction.
13. Place the gel in the vertical gel apparatus (reconfigured for
running) and add 1Â TBE to both buffer reservoirs. Remove
the comb carefully to avoid deformation of the wells. Pre-run
the gel for 20 min (15 (W)).
14. Before RNA loading, use a syringe to wash out all wells thoroughly, removing excess urea, and remove any air bubbles that
have accumulated under the gel.
15. Load the denatured RNA samples onto the polyacrylamide gel.
Avoid loading samples into the first and the last wells of the gel.
16. Perform PAGE at 3 W for 15–20 min, until the RNA samples
have entered the gel. Then, increase to 15 W for the rest of the
electrophoresis, which lasts about 1.5 h.
17. The pre-stained size marker allows one to follow the RNA
migration visually. The electrophoresis is usually stopped
400
Marcel Bo ¨ hrer et al.
the other samples for all remaining steps.
3. Freeze the samples at À20
C (overnight).
4. Dry the frozen samples completely by sublimation in a SpeedVac concentrator (45 min for a 20––30 μL sample volume
at RT).
5. Resuspend the samples in 12 μL RNA-loading buffer (see
Note 13).
6. Heat the samples to 95
C for 3 min, and then keep them
on ice.
7. After this RNA heat denaturation, add DynaMarker Prestain
“small RNA Plus” marker (5.5 μL) into the RNA oligo size
standard mix.
8. Set up the vertical gel apparatus for casting. Make sure that all
the components are clean.
9. Prepare a 16% polyacrylamide gel mixture in a 50 mL Falcon
tube, containing 11.1 g urea, 6.0 mL 5Â TBE, and 12.0 mL
acrylamide/bis-acrylamide (19:1). Fill with Milli-Q water up
to 30 mL and mix to dissolve all the components.
10. Rapidly add 30 μL TEMED and 300 μL 10% APS to the
mixture just before casting. Immediately cap and invert the
Falcon tube to mix. Then, without hesitation, steadily transfer
this solution between the glass plates of the gel apparatus using
a 10 mL pipette.
11. Add the comb while avoiding any air bubbles before the gel
starts to polymerize.
12. Let the gel sit for about 20–30 min for complete polymerization. Leftover gel mixture in the Falcon tube can be observed
as a reference for the polymerization reaction.
13. Place the gel in the vertical gel apparatus (reconfigured for
running) and add 1Â TBE to both buffer reservoirs. Remove
the comb carefully to avoid deformation of the wells. Pre-run
the gel for 20 min (15 (W)).
14. Before RNA loading, use a syringe to wash out all wells thoroughly, removing excess urea, and remove any air bubbles that
have accumulated under the gel.
15. Load the denatured RNA samples onto the polyacrylamide gel.
Avoid loading samples into the first and the last wells of the gel.
16. Perform PAGE at 3 W for 15–20 min, until the RNA samples
have entered the gel. Then, increase to 15 W for the rest of the
electrophoresis, which lasts about 1.5 h.
17. The pre-stained size marker allows one to follow the RNA
migration visually. The electrophoresis is usually stopped
400
Marcel Bo ¨ hrer et al.