in vitro Transcription
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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°C.
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.
