3.1.2 Quality Control of
ImpA
1. Dissolve 1 mg ImpA in 20 μL DMF. Draw a starting line on a
TLC plate. Spot 1 μL solution A, solution B, and ImpA/DMF
solution, respectively, on the starting line.
2. Use iso-propanol:water:25% ammonia (7:2:1) as solvent system and run the TLC in a chromatography chamber. Take out
the plate when the solvent front is about 0.3 cm away from the
top of the plate. Label the solvent front with a pencil.
3. Dry the plate and visualize the spots under 254 nm UV lamp.
The material on the starting line should completely disappear,
indicating the successful and efficient production of ImpA
(Fig. 2c).
3.1.3 Adenylation of 3
0
Adapter Oligonucleotides
(See Note 1)
1. Prepare 150 μL solution containing 100 mM ImpA
(MW ¼ 396.3) and 50 mM MgCl 2 .
2. Add 100 μL ImpA/MgCl 2 solution into 50 nM 5
0 phosphorylated 3
0 NH 2 -linker modified 3
0 adapter (Table 1) oligonucleotide dry powder (see Note 2). Incubate the solution at 50
C
for 1.5 h. Add the remaining 50 μL ImpA/MgCl 2 solution and
incubate another 1.5 h.
3. After reaction, reduce the volume to about 75 μL in a SpeedVac. Then add one volume (75 μL) loading buffer (8 M urea,
50 mM EDTA pH 8.0, 0.5 mg/mL bromophenol blue).
4. Prepare 150 mL 20% urea-polyacrylamide gel (Table 2) with
the size of 33 cm  40 cm  0.25 mm containing only one well
or two wells. Load the sample in one well and load bromophenol blue and xylene cyanol loading buffer in the other if two
wells are used. Run electrophoresis with 1Â TBE buffer for
3.5 h at 3500 mV until xylene cyanol is about to exit the
bottom of the gel.
5. Dismantle the gel and wrap it with plastic film. Place it on a
fluorescence-indicator-coated silica gel plate and visualize the
band with a 254 nm UV lamp. The intensity of two bands is
typically similar, indicative of the pre-adenylation of approximately half amount of the initial 5
0 phosphorylated 3
0 NH 2 -
linker modified 3
0 adapter oligonucleotide (Fig. 2d).
6. Mark the upper bands and excise the product band. Use a clean
blade to cut the gel along the sketch. Dice the gel into small
pieces and carefully transfer them to a 1.5 mL tube with 350 μL
0.4 M NaCl.
7. Tumble in the Eppendorf thermomixer at a speed of
1200 rpm/min at 4
C overnight to elute the adapter.
8. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble 30 min at 4
C.
Identification and Quantification of sRNAs
237
ImpA
1. Dissolve 1 mg ImpA in 20 μL DMF. Draw a starting line on a
TLC plate. Spot 1 μL solution A, solution B, and ImpA/DMF
solution, respectively, on the starting line.
2. Use iso-propanol:water:25% ammonia (7:2:1) as solvent system and run the TLC in a chromatography chamber. Take out
the plate when the solvent front is about 0.3 cm away from the
top of the plate. Label the solvent front with a pencil.
3. Dry the plate and visualize the spots under 254 nm UV lamp.
The material on the starting line should completely disappear,
indicating the successful and efficient production of ImpA
(Fig. 2c).
3.1.3 Adenylation of 3
0
Adapter Oligonucleotides
(See Note 1)
1. Prepare 150 μL solution containing 100 mM ImpA
(MW ¼ 396.3) and 50 mM MgCl 2 .
2. Add 100 μL ImpA/MgCl 2 solution into 50 nM 5
0 phosphorylated 3
0 NH 2 -linker modified 3
0 adapter (Table 1) oligonucleotide dry powder (see Note 2). Incubate the solution at 50
C
for 1.5 h. Add the remaining 50 μL ImpA/MgCl 2 solution and
incubate another 1.5 h.
3. After reaction, reduce the volume to about 75 μL in a SpeedVac. Then add one volume (75 μL) loading buffer (8 M urea,
50 mM EDTA pH 8.0, 0.5 mg/mL bromophenol blue).
4. Prepare 150 mL 20% urea-polyacrylamide gel (Table 2) with
the size of 33 cm  40 cm  0.25 mm containing only one well
or two wells. Load the sample in one well and load bromophenol blue and xylene cyanol loading buffer in the other if two
wells are used. Run electrophoresis with 1Â TBE buffer for
3.5 h at 3500 mV until xylene cyanol is about to exit the
bottom of the gel.
5. Dismantle the gel and wrap it with plastic film. Place it on a
fluorescence-indicator-coated silica gel plate and visualize the
band with a 254 nm UV lamp. The intensity of two bands is
typically similar, indicative of the pre-adenylation of approximately half amount of the initial 5
0 phosphorylated 3
0 NH 2 -
linker modified 3
0 adapter oligonucleotide (Fig. 2d).
6. Mark the upper bands and excise the product band. Use a clean
blade to cut the gel along the sketch. Dice the gel into small
pieces and carefully transfer them to a 1.5 mL tube with 350 μL
0.4 M NaCl.
7. Tumble in the Eppendorf thermomixer at a speed of
1200 rpm/min at 4
C overnight to elute the adapter.
8. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble 30 min at 4
C.
Identification and Quantification of sRNAs
237