3.7 Dephosphorylation
1. Prepare 1Â PNK buffer (pH 6.5) from 5Â stock.
2. Wash beads with 850 μL of ice-cold 1Â PNK buffer (pH 6.5)
by inverting several times.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 20 μL of PNK mix (4 μL 5Â PNK buffer
(pH 6.5), 0.5 μL T4 PNK, 0.5 μL RiboLock, 15 μL H 2 O).
5. Incubate for 20 min at 37
C at 1100 rpm in a thermomixer.
6. Wash beads twice with ice-cold 850 μL of PNK wash buffer
(pH 7.4) for 1 min by inverting several times.
7. Centrifuge for 1 min at 500 Â g and discard supernatant.
8. Wash beads once with 850 μL ice-cold high-salt buffer for
1 min by inverting several times.
9. Centrifuge for 1 min at 500 Â g and discard supernatant.
10. Wash beads twice with 850 μL of PNK wash buffer (pH 7.4)
for 1 min by inverting several times.
11. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
3.8 Linker Ligation
1. Prepare 1Â ligation buffer from 4Â stock.
2. Wash beads once with 850 μL of ice-cold 1Â ligation buffer for
1 min by inverting several times.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 20 μL ligation mix (9 μL H 2 O, 4 μL 4Â
ligation buffer, 1 μL RNA ligase, 0.5 μL RiboLock, 4 μL 50%
PEG8000).
5. Add 1.5 μL of pre-adenylated L3 adapter (20 μM).
6. Mix thoroughly and centrifuge briefly.
7. Incubate for at least 16 h at 16
C and 1100 rpm in a
thermomixer.
8. Add 500 μL PNK wash buffer (pH 7.4) and mix thoroughly.
9. Centrifuge for 1 min at 500 Â g and remove supernatant.
10. Wash beads twice with 850 μL of ice-cold high-salt buffer for
5 min under constant rotation.
11. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
12. Wash beads with 1 mL of ice-cold PNK wash buffer (pH 7.4)
for 5 min under constant rotation.
13. Centrifuge for 1 min at 500 Â g and discard supernatant.
14. Repeat washing step and leave second wash including beads
behind (do not centrifuge).
262
Tino Ko ¨ ster and Dorothee Staiger
1. Prepare 1Â PNK buffer (pH 6.5) from 5Â stock.
2. Wash beads with 850 μL of ice-cold 1Â PNK buffer (pH 6.5)
by inverting several times.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 20 μL of PNK mix (4 μL 5Â PNK buffer
(pH 6.5), 0.5 μL T4 PNK, 0.5 μL RiboLock, 15 μL H 2 O).
5. Incubate for 20 min at 37
C at 1100 rpm in a thermomixer.
6. Wash beads twice with ice-cold 850 μL of PNK wash buffer
(pH 7.4) for 1 min by inverting several times.
7. Centrifuge for 1 min at 500 Â g and discard supernatant.
8. Wash beads once with 850 μL ice-cold high-salt buffer for
1 min by inverting several times.
9. Centrifuge for 1 min at 500 Â g and discard supernatant.
10. Wash beads twice with 850 μL of PNK wash buffer (pH 7.4)
for 1 min by inverting several times.
11. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
3.8 Linker Ligation
1. Prepare 1Â ligation buffer from 4Â stock.
2. Wash beads once with 850 μL of ice-cold 1Â ligation buffer for
1 min by inverting several times.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 20 μL ligation mix (9 μL H 2 O, 4 μL 4Â
ligation buffer, 1 μL RNA ligase, 0.5 μL RiboLock, 4 μL 50%
PEG8000).
5. Add 1.5 μL of pre-adenylated L3 adapter (20 μM).
6. Mix thoroughly and centrifuge briefly.
7. Incubate for at least 16 h at 16
C and 1100 rpm in a
thermomixer.
8. Add 500 μL PNK wash buffer (pH 7.4) and mix thoroughly.
9. Centrifuge for 1 min at 500 Â g and remove supernatant.
10. Wash beads twice with 850 μL of ice-cold high-salt buffer for
5 min under constant rotation.
11. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
12. Wash beads with 1 mL of ice-cold PNK wash buffer (pH 7.4)
for 5 min under constant rotation.
13. Centrifuge for 1 min at 500 Â g and discard supernatant.
14. Repeat washing step and leave second wash including beads
behind (do not centrifuge).
262
Tino Ko ¨ ster and Dorothee Staiger