The paper describes a method for synthesizing small, defined-length RNAs using T7 RNA polymerase and synthetic DNA templates containing the T7 promoter. The study demonstrates that partially single-stranded templates, base-paired only in the -17 to +1 promoter region, are as active as linear plasmid DNA in transcription. Runoff transcripts initiate at a predictable position but may have one nucleotide more or less on the 3' terminus. In addition to full-length products, the reactions yield smaller oligoribonucleotides, likely due to abortive initiation events. Variants in the +1 to +6 region of the promoter reduce transcription efficiency but increase the variety of RNAs produced. The optimal conditions for transcription reactions have been optimized to allow the synthesis of milligram amounts of RNA fragments ranging from 12 to 35 nucleotides in length. The method offers advantages such as avoiding unwanted flanking sequences, not being limited by restriction sites, and eliminating the need for cloning and plasmid preparation. The study also explores the effects of promoter size and sequence on transcription efficiency, finding that the T7 RNA polymerase does not require the non-template strand in the -17 to +6 region. The method is scalable and can produce large quantities of RNA, making it suitable for biochemical and biophysical studies.The paper describes a method for synthesizing small, defined-length RNAs using T7 RNA polymerase and synthetic DNA templates containing the T7 promoter. The study demonstrates that partially single-stranded templates, base-paired only in the -17 to +1 promoter region, are as active as linear plasmid DNA in transcription. Runoff transcripts initiate at a predictable position but may have one nucleotide more or less on the 3' terminus. In addition to full-length products, the reactions yield smaller oligoribonucleotides, likely due to abortive initiation events. Variants in the +1 to +6 region of the promoter reduce transcription efficiency but increase the variety of RNAs produced. The optimal conditions for transcription reactions have been optimized to allow the synthesis of milligram amounts of RNA fragments ranging from 12 to 35 nucleotides in length. The method offers advantages such as avoiding unwanted flanking sequences, not being limited by restriction sites, and eliminating the need for cloning and plasmid preparation. The study also explores the effects of promoter size and sequence on transcription efficiency, finding that the T7 RNA polymerase does not require the non-template strand in the -17 to +6 region. The method is scalable and can produce large quantities of RNA, making it suitable for biochemical and biophysical studies.