3. Remove unincorporated [γ
32 P]-ATP with a Sephadex G-50
spin column. To prepare a G-50 column, add 2 mm of fiberglass into the bottom of a 1 mL syringe. Place the syringe into a
14 mL tube. Load swelling watered G-50 resin into the syringe
and allow the water to drop out (but do not let the resin dry).
Add resin until the syringe is filled. Centrifuge for 45 s at
2000 Â g. Place the syringe in a new 14 mL tube.
4. Add 50 μL of water to the PNK reaction mixture and load it
onto the top of the G-50 column. Centrifuge for 45 s at
2000 Â g to elute the radioactive probe. Discard the syringe
in the radioactive waste disposal container.
3.11 Northern
Hybridization
1. Wet the membrane with PerfectHyb™ Plus solution and place
it into a cylindrical glass hybridization tube. Make sure that the
membrane is well attached to the tube wall.
2. Add 4 mL of PerfectHyb™ Plus hybridization solution into
the tube.
3. Add 940 μL of PerfectHyb™ Plus to the radiolabeled probe,
and transfer it into the hybridization tube.
4. Place the tube into the hybridization oven, and incubate with
gentle rotation overnight at 42
C.
5. Discard the probe into the radioactive waste disposal container
or keep it at À20
C if you wish to reuse the probe another time
(the half-time of
32
P is 14 days).
6. Pour 10 mL of 2Â SSC into the tube and wash the membrane
for 10 min at 42
C with gentle rotation.
7. Discard the 2Â SSC buffer into the waste container dedicated
to radioactivity. Repeat step 6.
8. Discard 2Â SSC as above. Add 10 mL of 2Â SSC and 0.1% SDS
into the tube and wash the membrane for 30 min at 42
C with
gentle rotation.
9. Discard the washing solution. Place the membrane on a sheet
of Whatman paper to remove excess liquid. Then place the
membrane between two sheets of plastic wrap.
10. Expose the radioactive signal of membrane to a phosphorimager screen or to X-ray film. Image the screen using a phosphorimager or develop the X-ray film (see Note 16).
11. As an illustration, the autoradiography of a northern blot
showing the successful transfection of sncRNA into
A. thaliana protoplasts is presented Fig. 2. In this example,
an oligoribonucleotide (5
0 -ACCATCTGAGCTACATCCCC3
0 ) corresponding to a previously identified tRF Ala of 20 nt
(tRF Ala20) derived from the 5
0 extremity of an A. thaliana
cytosolic alanine tRNA [5] has been incorporated by the protoplasts. The oligoribonucleotide transfection was further
426
Ste ´ phanie Lalande et al.
32 P]-ATP with a Sephadex G-50
spin column. To prepare a G-50 column, add 2 mm of fiberglass into the bottom of a 1 mL syringe. Place the syringe into a
14 mL tube. Load swelling watered G-50 resin into the syringe
and allow the water to drop out (but do not let the resin dry).
Add resin until the syringe is filled. Centrifuge for 45 s at
2000 Â g. Place the syringe in a new 14 mL tube.
4. Add 50 μL of water to the PNK reaction mixture and load it
onto the top of the G-50 column. Centrifuge for 45 s at
2000 Â g to elute the radioactive probe. Discard the syringe
in the radioactive waste disposal container.
3.11 Northern
Hybridization
1. Wet the membrane with PerfectHyb™ Plus solution and place
it into a cylindrical glass hybridization tube. Make sure that the
membrane is well attached to the tube wall.
2. Add 4 mL of PerfectHyb™ Plus hybridization solution into
the tube.
3. Add 940 μL of PerfectHyb™ Plus to the radiolabeled probe,
and transfer it into the hybridization tube.
4. Place the tube into the hybridization oven, and incubate with
gentle rotation overnight at 42
C.
5. Discard the probe into the radioactive waste disposal container
or keep it at À20
C if you wish to reuse the probe another time
(the half-time of
32
P is 14 days).
6. Pour 10 mL of 2Â SSC into the tube and wash the membrane
for 10 min at 42
C with gentle rotation.
7. Discard the 2Â SSC buffer into the waste container dedicated
to radioactivity. Repeat step 6.
8. Discard 2Â SSC as above. Add 10 mL of 2Â SSC and 0.1% SDS
into the tube and wash the membrane for 30 min at 42
C with
gentle rotation.
9. Discard the washing solution. Place the membrane on a sheet
of Whatman paper to remove excess liquid. Then place the
membrane between two sheets of plastic wrap.
10. Expose the radioactive signal of membrane to a phosphorimager screen or to X-ray film. Image the screen using a phosphorimager or develop the X-ray film (see Note 16).
11. As an illustration, the autoradiography of a northern blot
showing the successful transfection of sncRNA into
A. thaliana protoplasts is presented Fig. 2. In this example,
an oligoribonucleotide (5
0 -ACCATCTGAGCTACATCCCC3
0 ) corresponding to a previously identified tRF Ala of 20 nt
(tRF Ala20) derived from the 5
0 extremity of an A. thaliana
cytosolic alanine tRNA [5] has been incorporated by the protoplasts. The oligoribonucleotide transfection was further
426
Ste ´ phanie Lalande et al.