3. Rnl2 K227Q (together with 3
0 ligation buffer).
4. 10Â BSA (Diluted 10 times with 100Â Ac-BSA).
5. 50% DMSO prepared in RNase-free water.
2.7 5
0 Ligation
1. The same materials as described in Subheading 2.6, except 3
0
adapter and Rnl2 K227Q.
2. 5
0 Adapter (Table 1).
3. Rnl1 (together with 5
0 ligation buffer).
4. 10 mM ATP prepared with RNase-free water.
5. SR-RT primer purified in Subheading 3.2.
2.8 Reverse
Transcription
1. PCR machine.
2. PCR tubes and all size tips.
3. Hydrion S/R Dispenser pH papers (pH 1.0–11.0).
4. RNase-free water.
5. Autoclaved double-distilled water.
6. 10 mM dNTP mix prepared with RNase-free water.
7. 0.1 M DTT prepared with RNase-free water.
8. SSII (together with Reverse transcription kit).
9. RNase inhibitor (RNase OUT).
10. 150 mM KOH/20 mM Tris-Base.
11. 150 mM HCl.
2.9 PCR
1. Gel electrophoresis casts, trays, combs, tank, and power device.
2. PCR tubes and all size tips.
3. Autoclaved double-distilled water.
4. 2 mM dNTPs.
5. SR primer and 3
0 primers purified from Subheading 3.2
(Table 1).
6. cDNA synthesized from Subheading 3.8.
7. KOD hot start DNA polymerase (together with kit).
8. Agarose.
9. 0.5Â TBE buffer.
10. Ethidium bromide (EB).
11. 1 kb DNA ladder.
12. Phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0.
13. Chloroform.
14. 3 M NaAc.
15. Ethanol.
Identification and Quantification of sRNAs
233