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Summary: 85
RNA Extraction and Labeling
1. To IP pellet (~ 25 µl vol), add 175 µl of: 10 mM HEPES-NaOH, pH 7.5
300 mM NaCl
1 mM EDTA
0.2% SDS
10 µg glycogen (molecular biology grade)
250 µg proteinase K (from 25 mg/ml stock)
2. Incubate 15 minutes at 37°C. Vortex every 2-3 minutes.
3. Extract twice with an equal volume of ØOH:CHCl3. Vortex 30 seconds and spin for 30 seconds for
each extraction. Avoid transfer of interface layer after extraction.
4. Ethanol precipitate with 3 volumes of 100% EtOH. No need to add 3 M NaOAc. 1 hour at 20°C.
Spin 30 minutes at 4°C.
5. Wash with DEPC-treated 75% EtOH.
6. Dry in air or using Speed-Vac.
7. Labeling 3' ends. To each pellet, add 10 µl containing: 1 X NEB RNA Ligase buffer
10% DMSO (ultrapure)
0.1 mg/ml BSA (NEB)
10 units RNase inhibitor (0.25 µl RNasin)
10 units NEB RNA Ligase (0.5 µl)
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