in vitro Transcription Reaction 1 µl 10X Transcription Buffer (Ambion) 1 µl 10X NTPs (4 mM ATP, CTP, 1 mM GTP, UTP) 2 µl 10 mM GpppG cap (Pharmacia) 2 µl a[32P]-UTP (NEN) 800 Ci/mmol 0.2 µl RNasin (Promega) 1 µl 0.1M DTT 1.8 µl H2O 0.5 µl Linearized Transcription Template (1 µg/µl) 0.5 µl Polymerase (SP6/T7/T3) Incubate for 1 hour at 37℃. Add 10 µl STOP solution (formamide loading buffer) to each reaction, boil, and load onto a pre-run 5% denaturing polyacrylamide gel. Run desired distance. Cut out bands and soak in 500 µl RNA elution buffer (300 mM NaOAc, pH 6.1, 0.2% SDS, 1 mM EDTA) for 1 hour - overnight Precipitate RNA with 2 volumes ethanol. Resuspend in 20 µl H2O and quantitate 0.5 µl. |
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