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°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.