2.1.3 Adenylation of 3
0
Adapter Oligonucleotides
1. SpeedVac.
2. Glasses, combs, and clips used for gel preparation.
3. Gel electrophoresis tank and power device.
4. 1.5 mL Eppendorf tubes and all size tips.
5. Plastic film.
6. UV lamp and silica gel plates.
7. Blades.
8. Nano drop.
9. 50 mM MgCl 2 dissolved in sterilized deionized water.
10. 5
0 Phosphorylated 3
0 NH 2 -linker modified 3
0 adapter (Table 1)
oligonucleotide dry powder.
11. Autoclaved double-distilled water.
12. Diethylpyrocarbonate (DEPC).
13. RNase-free water (1 L water mix well with 1 mL DEPC.
Incubate the mix at room temperature overnight in a hood.
Autoclave treated water at 121
C for 20 min to
destroy DEPC).
14. Urea-polyacrylamide gel reagents: UreaGel concentrate solution (National diagnostics), UreaGel diluent solution
(National diagnostics), UreaGel buffer (National diagnostics),
TEMED, 10% ammonium persulfate (APS) (10 g in 100 mL
water) (Table 2).
15. 1Â TBE buffer diluted from 10Â TBE buffer: 1 M Tris base,
1 M boric acid, 0.02 M EDTA (disodium salt) prepared with
RNase-free water.
16. Loading buffer: 8 M urea, 50 mM EDTA pH 8.0, 0.5 mg/mL
bromophenol blue.
Table 1
Markers, adapters, and primers
Product
Index primer sequence
19 nt size
marker
5
0 -rCrGrUrArCrGrCrGrGrGrUrUrUrArArArCrGrA-3
0
24 nt size
marker
5
0 -rCrGrUrArCrGrCrGrGrArArUrArGrUrUrUrArArArCrUrGrU-3
0
SR Primer
5
0 -AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGs-A-3
0
SR-RT Primer 5
0 -GCCTTGGCACCCGAGAATTCCA-3
0
3
0 adapter
5
0 -NNNNTGGAATTCTCGGGTGCCAAGG-NH 2 -3
0
5
0 adapter
5
0 -rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3
0
Identification and Quantification of sRNAs
229