Prepare the linearized template DNA To prepare template from plasmid DNA

a. Linearize 5-20 pg of plasmid DNA by digestion with a fivefold excess of an appropriate restriction enzyme that cleaves either within the cloned DNA sequence or downstream from the DNA sequence. The distance from the promoter to the newly created terminus should be 200-400 bp. Make sure not to use an enzyme that separates the promoter from the sequence of interest. Because bacteriophage-encoded RNA polymerases may initiate transcription at 3'-protruding termini, choose a restriction enzyme that generates a blunt terminus or a 5' extension.

b. At the end of the digestion, analyze an aliquot (approx. 200 ng) of the reaction by agarose gel electrophoresis. No trace of circular plasmid DNA should be visible. If necessary, add more restriction enzyme and continue digestion until no more circular plasmid DNA can be detected.

c. Purify the linear DNA by extracting twice with phenol:chloroform and then recover the DNA by standard precipitation with ethanol. After washing the precipitate with 70% ethanol, dissolve the DNA in TE (pH 7.6) at a concentration of 1 pg/pl.

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