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KAI-OLAF NETZER
• T7 RNA polymerase
• Rnasin™ 20 U/fll
• DTT, 0.2 M
• DNase I, RNase-free, 1mg/ml
• tRNA (yeast, DNase-free, RNase-free; e.g. Sigma) 1 flglfll, which works as
carrier in the precipitation reaction
• Ammonium acetate 5 M
• Ethanol 100%, 70%
Procedure
ln advance 1. Linearize the DNA template with the appropriate restriction enzyme.
2. Purify the DNA by using a chaotropic buffer/silica-gel spin column system (e. g. Qiagen).
3. Check an aliquot (0.1-0.2 )lg) ofthe DNA digest by agarase gel electrophoresis.
4. Dilute the DNA to 0.1 pmol/fll (i.e. 200 ng/fll for a 3 kb plasmid) in DEPC
treated water.
ln vitro 5. At room temperature, mix: the following components in a reaction tube:
transcription
DNA template
2 f..ll
0.2 pmol
10x buffer
2 f..ll
rNTP mix (w/o rUTP)
2 f..ll
DTT
1 f..ll
0.2 f..lmol
RnasinTM
1 f..ll
DEPC water
T7 RNA polymerase
1 f..ll
[ 35 S]rUTP
1 f..ll
12.5 f..lCi
Note: If RNA is to be synthesized for in vitro translation or as control in
cDNA reactions, no radiolabeling is necessary. Replace the [3 5 S]rUTP
with nonradioactive rUTP in the rNTP mix: (5 mM). If working with radio-