Linearizing plasmid (can be scaled up or down as needed):
50 µl:
5 µl 10x buffer
20 µg plasmid
40 units restriction enzyme
37C, ≥2h.
150 µl Te
5 µl 0.2M EDTA
Extract with PCA.
20 µl 3M NaAc (pH6.0)
500 µl EtOH
Ice, ≥10 min
14,000 rpm, 4C, 30 min
Remove EtOH
Wash w. 70% EtOH
Dry
Dissolve in Te to ≈1 µg/µl

Transcription (can be scaled up or down as needed):
20 µl:
2 µl 10xT7
2 µl 50 mM DTT
2 µl 10 mM NTPs
2 µg linearized plasmid
4 µl T7 RNA polymerase
37C, 1-2h.
20 µl Formamide load buffer
95C, 30 sec.
Run on PAGE/6M urea
Visualize RNA by UV shadowing and cut out band.
Transfer gel piece to 400 µl extraction buffer
Nutate at RT over night.
Transfer extraction buffer to new eppendorf tube
Extract with PCA
1 ml EtOH
Ice, ≥10 min
14,000 rpm, 4C, 30 min
Remove EtOH
Wash w. 70% EtOH
Dry
Dissolve in autoclaved millipore water
Measure concentration and purity by A260/A280

Buffers:
10xT7:
400 mM Tris-HCl pH 8.0
60 mM MgCl2

Formamide load buffer:
Deionized formamide
5 mM EDTA
0.1% Bromophenol blue
0.1% Xylene cyanol

Extraction buffer:
300 mM NaAc pH 6.0
5 mM EDTA
0.25% SDS